Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection

In this study we investigated whether morphology and chromatinanomalies in human spermatozoa can influence fertilization afterintracytoplasmic sperm injection (ICSI). We examined unfertilizedoocytes, using the fluorochrome Hoechst 33342, to determinewhether a relationship exists between failure of fertilizationand sperm chromatin quality. Sperm chromatin packaging qualitywas assessed using the chromomydn A3 (CMA3) fluorochrome, andthe presence of DNA damage in spermatozoa, using in-situ nicktranslation. Normal males present sperm parameters with a normalmorphology of >20%, CMA3 fluorescence of <30% and exhibitendogenous nicks in <10% of their spermatozoa. When patientswere separated according to these values no difference was observedin their fertilization rates after ICSL When the unfertilizedICSI oocytes were examined, we found that patients with CMA3fluorescence of <30% and nicks in <10% of their spermatozoahad only 17.5 and 21.6% respectively of their unfertilized oocytescontaining spermatozoa that remained condensed. In contrast,patients with higher CMA3 and nick values had a significantlyhigher number, 412 and 48.9%, of their unfertilized oocytescontaining condensed spermatozoa. Sperm morphology did not showany such pattern. The percentage of spermatozoa which had initiateddecondensation in unfertilized oocytes was not influenced bymorphology, CMA3 fluorescence or nicks. In light of these resultswe postulate that poor chromatin packaging and/or damaged DNAmay contribute to failure of sperm decondensation after ICSIand result in failure of fertilization.     

Cytogenetic abnormalities of unfertilized oocytes generated from in- vitro fertilization and intracytoplasmic sperm injection: a double- blind study

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.      

Gamete-specific DNA fragmentation in unfertilized human oocytes after intracytoplasmic sperm injection

The objective of the present study was to assess the integrity of maternal and/or paternal chromatin in injected oocytes that remained unfertilized after intracytoplasmic sperm injection (ICSI). The study was performed on 102 oocytes that failed to show pronuclear formation 18-20 h after ICSI. We used chromatin labelling with 4,6-diamidino-2- phenylindole (DAPI) to identify maternal and paternal chromatin, coupled with biotin-mediated end-labelling to assess DNA fragmentation in each gamete. It was shown that 50% of oocytes without pronuclear formation following ICSI contained chromatin with damaged DNA, and that the source of the DNA fragmentation was equally divided between the spermatozoon (25.8%) and the oocyte (24.4%). A significantly greater proportion of condensed spermatozoa in human oocytes had damaged DNA, compared to decondensed spermatozoa (24.7 compared to 5.9%, P=0.002). There was a significant increase in the incidence of DNA fragmentation in oocytes from patients older than 35 years (65+/-1.2%) compared to those from younger patients (36+/-1.0%) (P < 0.05). Further, 17% of unfertilized oocytes contained no paternal chromatin. Thus, DNA fragmentation in both spermatozoa and oocytes is associated with failure of fertilization in ICSI. In some cases of severe male factor infertility, a significant proportion of spermatozoa injected into oocytes may contain fragmented DNA. Injection of oocytes with spermatozoa containing abnormal chromatin will probably result in failure of sperm decondensation and fertilization. In older women, a significant proportion of oocytes injected may contain fragmented DNA. These observations may explain the consistent inability of most clinics to achieve fertilization rates higher than 65% with ICSI.      

Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa

This study aimed to investigate the association between anomaliesin sperm chromatin packaging, morphology and fertilization inpatients undergoing routine in-vitro fertilization (IVF) orsubzonal insemination (SUZI). Sperm chromatin packaging wasassessed using chromomycin A3 (CMA3), a fluorochrome specificfor guanine-cytosine rich sequences of DNA. One hundred to 150sperm cells were assessed in 55 patients to compare sperm chromatinpackaging and morphology to fertilization after IVF or SUZI.When the morphology and CMA3 fluorescence of individual spermatozoawas assessed, >75% of the macrocephalic sperm fluorescedin all patients. In contrast a mean of 37% of the spermatozoawith normal morphology fluoresced in IVF patients compared with58% of the normal spermatozoa in male factor patients treatedby SUZI. SUZI patients displaying a high fluorescence (>70%)in their spermatozoa also had a significantly lower fertilizationrate. Lower packaging quality in morphologically normal spermatozoamay represent a major limiting factor in the fertilizing abilityof male factor patients. This study confirms that a high percentageof CMA3 positivity is present in certain forms of male factorinfertility and that such a test may be used to distinguishseparate populations in morphologically normal spermatozoa. chromatin/chromomycin A₃/in-vitro fertilization/male infertility/spermatozoa     

Fertilization and early embryology: Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes

This study was undertaken to establish baseline data on thechromosomal status of ‘failed-fertilized’ oocytesderived from in-vitro fertilization (IVF) or intracytoplasmicsperm injection (ICSI) procedures. A cytogenetic analysis wasundertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162)of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphaseII (Mil) plates, of which 50.4% of the IVF and 47.5% of theICSI oocytes were analysed further. Chromosomes of the G-group(21–22) were identified with the majority of the anomalies.No overall significant difference in the aneuploidy rate wasfound for the IVF (37.3%) or ICSI (31.6%) oocytes, or with maternalage. However, chromosome anomalies, e.g. diploidy, fragmentedand broken chromatids, single sperm and oocyte chromatids, werefound in oocytes from IVF patients aged >36 years and inthe ICSI oocytes throughout the maternal age range (31–38years). The status of the polar body chromatin indicated thatthere was no overall significant difference in the maturationof the IVF and ICSI oocytes. Evidence of successful sperm deliverywas found in 72.5% (37/51) of the ICSI failed-fertilized oocytes.In this group there was a significant increase in the incidenceof premature chromosome condensation: 19.6% (10/51) containedsperm chromosomes, 7.8% (4/51) had swollen sperm heads, andthe remaining 45.0% had condensed sperm heads. The presenceof both sperm and Mil oocyte chromosomes was found in 19.6%(10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilizedoocytes. Specific fluorescent in-situ hybridization DNA probeswere used to re-analyse the chromosomes of karyotyped ‘failed-fertilized’IVF oocytes and, for the first time, applied to the karyotypedchromosomes of failed-fertilized ICSI oocytes. The hybridizationefficiency was 86–95% for the centromere probe and 100%for probes 21 and 18.     

Should ICSI be the treatment of choice for all cases of in-vitro conception?

The objective of this study was to examine different clinical scenarios of in-vitro conception, viz. fertilization with conventional IVF, IVF with high insemination concentration (HIC) and intracytoplasmic sperm injection (ICSI), and assess on a sibling oocyte comparison the hypothesis that ICSI should be performed in all cases requiring in-vitro conception. ICSI with husband's spermatozoa had a higher incidence of fertilization as compared with IVF or IVF with HIC with donor spermatozoa (if previous failure of fertilization had occurred) for unexplained infertility. Similarly, ICSI with husband's spermatozoa had as high an incidence of fertilization as IVF with donor spermatozoa for patients with severe oligozoospermia, asthenozoospermia and/or teratozoospermia, even when the spermatozoa were not selected for their morphology. Two studies were performed to assess ICSI in potential oocyte-related failure of IVF, viz. when fertilization occurred in >50% of oocytes for one group of patients, and in <50% of oocytes in a second group. In both of these studies a significant proportion of the oocytes that failed to fertilize with conventional IVF eventually fertilized after ICSI. The overall conclusion was that ICSI as a first option offers a higher incidence of fertilization, maximizes the number of embryos and minimizes the risk of complete failure of fertilization for all cases requiring in-vitro conception. However, among other concerns, current knowledge of ICSI as an outcome procedure does not provide the confidence to use this process in all cases of IVF for the time being.     

Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome

A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been suggested. The DNA content of spermatozoa might be responsible for inducing alterations in sperm morphology. The final nuclear shape, which is species-specific, depends on chromatin condensation during spermatogenesis as well as a precise organization of DNA within the nucleus. Many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility. Chromatin condensation is achieved by gradual substitution of lysinerich somatic histones by testis-specific histone and finally by protamine. In this study two groups of patients were compared: the first consisted of 63 patients who had undergone intracytoplasmic sperm injection (ICSI) with freshly ejaculated spermatozoa whereas the second included 47 patients assigned to ICSI with testes biopsy-extracted spermatozoa. In both groups chromatin condensation was assessed by aniline blue staining and morphology evaluated according to strict criteria. The condensed chromatin and morphology of spermatozoa were significantly (P < 0.0001) less in the second group compared to the first. However the fertilization, cleavage, implantation and pregnancy rates were almost the same in both investigated groups. There was no significant difference between the two groups with respect to ICSI outcome. The percentage of chromatin condensation (nuclear maturity) and morphologically-normal spermatozoa were significantly higher (P < 0.0001) in the ejaculated spermatozoa than in those from testis biopsy but the ICSI outcome (fertilization, cleavage, implantation and pregnancy rates) was the same. In view of these results the fertilization capability and the embryo quality obtained using testis biopsy extracted spermatozoa is not influenced by chromatin condensation and sperm morphology in testicular sperm extraction (TESE)-ICSI programmes. Therefore, it could be said that neither chromatin condensation nor morphology of testis extracted sperm could predict the fertilization, implantation and pregnancy rate in TESE-ICSI programmes.     

Ca2+载体A23187对受精失败人类成熟卵母细胞的补救激活作用

目的:观察Ca²⁺载体A23187对受精失败人类成熟卵母细胞的补救激活及激活后的胚胎发育情况。方法:收集常规体外受精(IVF)及卵浆内单精子注射(ICSI)后24h仍无受精征象的成熟卵母细胞作为研究对象,5μmol/LCa²⁺载体A23187中处理5min,观察第二极体排出及原核形成情况,激活后卵母细胞在体外继续培养2d。结果:IVF组及ICSI组卵母细胞激活率分别为64.9%(24/37)和73.2%(30/41)。ICSI组受精失败卵母细胞激活后主要表现为二极体二原核(2PB+2PN)(80%,24/30),而IVF组仅有20%的被激活卵母细胞表现为2PB+2PN,两组间差异具极显著(P＜0.01)。31个2PN卵母细胞继续培养,25个发生分裂,11个发育到2-4细胞,8个发育到4-8细胞,6个发育到8-细胞以上阶段。结论:Ca²⁺载体A23187能够有效地激活ICSI后受精失败卵母细胞恢复受精并继续发育成胚胎。     

A comparison of ICSI outcomes with fresh and cryopreserved epididymal spermatozoa from the same couples

The published experience with frozen-thawed epididymal spermatozoa and intracytoplasmic sperm injection (ICSI) suggests that fertilization and pregnancy success rates are comparable to those achieved with freshly retrieved spermatozoa. However, no study has exactly compared clinical outcomes between the two IVF/ICSI cycles in the same couples. To formally address this issue, we assessed ICSI outcomes in couples each of whom had had two IVF/ICSI cycles: one using fresh and the second using frozen-thawed epididymal spermatozoa obtained from a single aspiration procedure. From a pool of 101 consecutive patients undergoing IVF/ICSI with epididymal spermatozoa, 19 couples initially used fresh epididymal spermatozoa and subsequently underwent a second IVF/ICSI procedure with frozen-thawed spermatozoa from the same aspiration. Normal (2PN) oocyte fertilization rates, embryo quality and pregnancy rates were compared between the two IVF/ICSI cycles for each couple. In the fresh epididymal sperm group, 58.4% of the injected oocytes fertilized normally compared with 62.0% of the injected oocytes in the frozen-thawed epididymal sperm group, revealing no statistically significant difference. Graded embryo quality also did not differ significantly between the paired IVF/ICSI cycles. The clinical pregnancy rates were 31.6% (6/19) and 36.8% (7/19) in the first and second cycles respectively. All but one pregnancy were singletons. In summary, this study provides strong evidence to support the notion that motile, cryopreserved and thawed epididymal spermatozoa are equal to freshly retrieved spermatozoa for ICSI in couples with obstructive azoospermia.     

Andrology: Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection

In this report, we present the results of our first 100 consecutivecycles of intracytoplasmic sperm injection (ICSI). Overall,fertilization occurred in 98% of cycles and embryos were transferredin 94% (2.6 embryos per cycle). About 50% of patients had embryosfrozen. The overall fertilization rate was 71%, of which 4%were abnormally fertilized (three pronuclei). A total of 30clinical pregnancies were established (32% per transfer), resultingin 18 singleton, six twin and one triplet ongoing pregnancies.The implantation rate per embryo was 15%. There were no significantdifferences in the fertilization or pregnancy rates betweenpatients Who had only occasional motile spermatozoa in the ejaculate,semen that was too poor for routine in-vitro fertilization (IVF),or who had failed routine IVF and/or subzonal sperm injection(SUZI). A group of 18 patients were treated with both ICSI androutine IVF on their first cycle because of the high likelihoodof failed fertilization due to poor sperm morphology (<20%normal). In this group, ICSI oocytes had a fertilization rateof 76% compared to only 15% for the routine IVF (control) oocytes,and six patients conceived after transfer of ICSI embryos (33%),indicating that ICSI can be used successfully on 50% of theoocytes if fertilization failure is expected. Similarly, patientswho had failed to become pregnant with SUZI achieved excellentresults after ICSI. There were no significant differences betweenICSI and routine IVF in the proportions of grade 1, 2 or 3 embryoson day 3 post-oocyte recovery. In conclusion, we have achievedresults comparable to those reported from Belgium and we havefound that ICSI is universally applicable to all forms of severemale factor infertility. ICSI produces fertilization, pregnancyand freezing rates comparable to routine IVF with normozoospermicsamples and has none of the drawbacks of other assisted fertilizationtechniques.     

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm- oocyte interaction

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.      

Easily decapitated spermatozoa defect: a possible cause of unexplained infertility.

We report the first 16 cases of a new sperm abnormality which we call 'easily decapitated spermatozoa defect'. This was discovered during intracytoplasmic sperm injection (ICSI) in couples with unexplained infertility. Semen analysis was normal, but minimal micromanipulation for ICSI resulted in decapitation of the spermatozoon during immobilization. For some oocytes the head and tail were injected separately, in others the intact sperm was injected after minimal immobilization. A fertilization rate of 47.5% was obtained using ICSI. Conventional in-vitro fertilization (IVF) on sibling oocytes (three cases) or in a previous cycle (three cases) resulted in total failure of fertilization. All patients reached the embryo transfer stage and three pregnancies resulted. Findings on electron microscopy in four cases included spermatozoa with degeneration or absence of the basal plate, abnormalities of the proximal centriole and degeneration of the midpiece with a large cytoplasmic droplet. We conclude that an occult sperm abnormality presenting as easily decapitated spermatozoa during ICSI could be a cause of unexplained infertility, as it resulted in total failure of fertilization in conventional IVF. Further research is necessary to investigate this sperm abnormality.     

Cytogenetic analysis of unfertilized oocytes following intracytoplasmic sperm injection using spermatozoa from a globozoospermic man

A man with globozoospermia was treated in our in-vitro fertilization- intracytoplasmic sperm injection (ICSI) programme. In the treatment cycle, 24 oocytes were collected from his wife. All the oocytes were at metaphase II stage. The semen sample produced on the day had a normal sperm count, good motility, but with 100% globozoospermia. All oocytes were injected with randomly selected spermatozoa and of these, two oocytes showed two pronuclei and another contained a single pronucleus. The remainder were unfertilized. The normally fertilized oocytes (two pronuclear) cleaved to the four-cell stage and were transferred to the patient. At 48 h after ICSI, the 21 unfertilized oocytes were processed for cytogenetic analysis. All oocytes contained a haploid chromosome set. The only abnormality seen was a chromosome fragment in one metaphase. Eighteen oocytes contained decondensed sperm nuclei and of these, 14 nuclei were beginning to show signs of premature chromatin condensation (PCC) and the other four showed strong signs of PCC. Thus it appears that in some forms of globozoospermia, arrest of nuclear decondensation and/or PCC are another cause of fertilization failure. The most likely cause for this is the absence or down-regulation of spermatozoa associated activating factor in round-headed spermatozoa.      

Fertilization and early embryolgoy: Human oocyte activation following intracytoplasmic injection: the role of the sperm cell

The aim of this study was to investigate whether the human spermatozoonparticipates in the activation of human oocytes following intracytoplasmicsperm injection (ICSI) and if so, by what mechanism. In thefirst series of experiments, we randomized human oocytes whichhad remained unfertilized after in-vitro fertilization (IVF)or ICSI, for intracytoplasmic injection with live spermatozoa,spermatozoa presumed to be dead and no spermatozoa. Secondly,unfertilized human oocytes and freshly ovulated mouse oocyteswere randomized for intracytoplasmic and sub-zonal injectionwith human sperm cytosolic fraction (CF) before and after heattreatment. We found that oocyte injection with initially motilespermatozoa induces human oocyte activation at a significantlyhigher rate than injection with dead spermatozoa (61 versus0%; P < 0.001) or injection without a spermatozoon (61 versus14%; P < 0.001). Intracytoplasmic injection of CF activatedboth human and mouse oocytes at the same rate as sperm injectionof human oocytes (activation rates of 70 and 65% respectively).This effect was greatly reduced by heat treatment of the CF.From these experiments we conclude firstly that the human spermatozooninjected intracytoplasmically contributes to human oocyte activationand secondly that the spermatozoon releases into the oocytea heat-sensitive, intracellularly active factor, which is notspecies-specific.     

Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes

Among the possible mechanisms of oocyte activation after sperm penetration, it appears most likely that a protein released by the spermatozoon elicits a calcium elevation in the ooplasm. To further test this idea, cytosolic factors obtained from human spermatozoa by two different methods, freezing-thawing and sonication, were injected into mouse oocytes following which intracellular calcium release was measured. Of a total of 42 mouse oocytes, a pattern of calcium oscillations was observed in nine out of 16 oocytes injected with sonicated fraction, in all of eight oocytes with the frozen-thawed fraction and in none of 18 control oocytes. Injection of the frozen- thawed fraction also produced regular calcium oscillations in all of five in-vitro matured human oocytes. To assess the putative factor's ability to support fertilization, human oocytes that were not activated by prior intracytoplasmic injection of spermatozoa (ICSI) and round spermatids were reinjected with the frozen-thawed sperm fraction. Of 23 human oocytes which remained unfertilized after ICSI, 19 became activated after injection with sperm cytosolic factor; eight showed two pronuclei, three one pronucleus and eight showed three or more pronuclei. Of 11 oocytes unfertilized after prior round spermatid injection, two developed two pronuclei, four developed one pronucleus and two had three or more pronuclei. Cytogenetic analysis by fluorescence in-situ hybridization confirmed the existence of a male pronucleus in eight out of nine such zygotes displaying two or more pronuclei. Thus, human sperm extracts activated mouse and human oocytes after injection, as judged by calcium flux patterns in conjunction with male pronucleus formation.      

常规IVF受精失败补救ICSI后行冻融胚胎移植的临床价值

目的探讨冻融胚胎移植在常规体外受精(IVF)失败后补救卵胞浆内单精子注射(L-ICSI)中的应用价值。方法在12个常规体外受精失败周期中应用ICSI对未受精的MⅡ期卵子进行显微授精,将获得的优质胚胎进行冷冻,再择期行冻融胚胎移植。结果对93个未受精的MⅡ卵子接受L-ICSI,受精63枚,受精率为67.7%(63/93),异常受精3枚(2枚1PN,1枚3PN),57个正常受精卵发生卵裂,卵裂率为95.0%(57/60),优质胚胎率为43.9%(25/57),10例患者冷冻胚胎25枚,其中4例采用程序化冷冻,6例采用玻璃化冷冻。9个患者行冻融胚胎移植,共移植胚胎18枚(其中解冻后胚胎碎裂死亡5枚),其中1个周期因冻融后2个胚胎碎裂放弃移植,2例获得临床妊娠,1例分娩出正常婴儿,1例正在妊娠中,临床妊娠率为22.2%。结论 ICSI可使常规体外受精失败的卵子再受精,冻融胚胎移植可以解决胚胎与子宫内膜不同步的问题,获得相对满意的临床结局,具有一定的应用价值。     

Successful fertilization and pregnancy following ICSI and electrical oocyte activation.

In a total of 1048 intracytoplasmic sperm injection (ICSI) cycles, motile spermatozoa from four out of 424 patients (0.9%) failed to fertilize oocytes, despite an apparently successful ICSI procedure. No activation was observed in these injected oocytes. The spermatozoa from three of the four patients were injected into unfertilized mouse oocytes by ICSI (mouse test) to evaluate their oocyte activating ability. The oocyte activation rate of the spermatozoa of patients A, B, and C in the mouse test was 46, 100, and 86% respectively (control: 100%). Simultaneous injection of two spermatozoa from patient A into the mouse oocytes increased the oocyte activating rate to 89% (sham control: 29%). 100% fertilization rates were obtained for patients A and B by combining ICSI and electrical stimulation, and this resulted in pregnancy and the birth of healthy twins for the partner of patient A. Thus, it is considered that the spermatozoa of these patients are not lacking sperm factors but that the activity of these factors is depressed. The combination of ICSI and electrical stimulation is effective in these cases.     

Fate of the acrosome in ooplasm in pigs after IVF and ICSI

BACKGROUND: ICSI bypasses the sperm-oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. METHODS: In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. RESULTS: In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. CONCLUSIONS: The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.     

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.     

Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.