Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection

The aim of this study was to examine the relationship between different preincubation periods of oocytes and the outcome of intracytoplasmic sperm injection (ICSI). We analysed retrospectively 95 ICSI treatment cycles performed to alleviate severe male-factor infertility. Oocyte collection was performed approximately 36 h after human chorionic gonadotrophin administration. The cumulus-corona-oocyte complexes obtained were incubated until the moment of ICSI. Fertilization, embryo development and implantation rates were analysed in four groups, which were divided according to the time lapse between oocyte retrieval and ICSI: group I, < or =3 h (18 cycles); group II, >3-< or =6 h (52 cycles); group III, >6-< or =9 h (14 cycles); and group IV, >9-< or =12 h (11 cycles). Immediately before ICSI the cumulus and corona cells were removed from the oocytes. A total of 723 metaphase II oocytes were injected: 126 from group I, 380 from group II, 126 from group III and 91 from group IV. The fertilization rates obtained were 52.3, 66.8, 65.1 and 69.2% respectively [P < 0.05 (using the chi2 test) between group I and groups II, III and IV]. Cleavage rates were similar in all groups (68.1, 69.7, 79.2 and 79.3% respectively), but the proportion of good quality embryos (< or =20% fragmentation) was significantly lower (P < 0.05) in group I (24.2%) compared with groups II (39.8%) and IV (39.6%). However, no statistically significant differences were observed between the four groups with regard to implantation rates (11.7, 13.2, 10.4 and 20.4% respectively). The results suggest that a preincubation period between oocyte retrieval and ICSI can improve the fertilization rate and embryo quality. This period might be necessary for some oocytes to reach full cytoplasmic maturity, leading to a higher activation rate upon microinjection.      

Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection

The fertilization rates and further development of 528 humanmetaphase IT oocytes directly injected by a single spermatozoonwere analysed with respect to their morphological features atthe light microscopy level at the time of retrieval. The deviationsof oocyte morphology which were most frequently observed, afterremoval of cumulus cells, were dark incorporations, dark zonapellucida, large peri-vitelline space, spots, vacuoles, refractilebodies and irregular shape. These deviations correlated neitherwith the fertilization rate nor with the embryo quality score,as compared to ‘ideal’ oocytes. Since the majorityof oocytes displayed deviations from the ‘ideal’morphotype but were still fertilized and developed in cultureat a normal rate, they were probably as normal as ‘ideal’oocytes. Since some of these morphotypes, such as refractilebodies, have been shown to be associated with failure of fertilization,it seems that intracytoplasmic sperm injection may be an appropriatemethod of treatment for couples in whom repeated failure ofin-vitro fertilization is associated with the retrieval of dysmorphicoocytes in the presence of normal semen characteristics.     

Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection

In this study, we compared the fertilization rate and embryo quality after intracytoplasmic sperm injection (ICSI) as they relate to oocyte morphology. A total of 654 ICSI cycles yielding 5903 metaphase II oocytes were observed. The oocytes retrieved in these cycles were divided into (i) normal oocytes, (ii) oocytes with extracytoplasmic abnormalities (dark zona pellucida and large perivitelline space), (iii) oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, and refractile body), (iv) oocytes with shape abnormalities, and (v) oocytes with more than one abnormality (double and triple abnormalities). Intracytoplasmic vacuoles and aggregates of smooth endoplasmic reticulum were not recorded separately. The fertilization rate and quality of morphologically graded embryos did not differ between the groups. There were 77 cycles where all transferred embryos were derived from abnormal oocytes, and 164 cycles where all embryos were derived from normal oocytes. These cycles were studied further. The two groups were comparable regarding mean female age, duration of infertility, duration of ovarian stimulation, number of ampoules of gonadotrophin injected, and number of oocytes retrieved. Two clinical pregnancy rates (44.4 versus 42.1%) and implantation rates per embryo (10.3 versus 13.2%) were similar. In conclusion, in couples undergoing ICSI, abnormal oocyte morphology is not associated with a decreased fertilization rate or unfavourable embryo quality. Furthermore, embryos derived from abnormal oocytes yield similar clinical pregnancy and implantation rates when transferred compared with embryos derived from normal oocytes.      

Human oocyte activation after intracytoplasmic sperm injection

The concentration of acetoxymethyl ester of fluo-3 given inthe subsection ‘Measurement of [Ca²⁺]_i’ of the ‘Materialsand methods’ on page 512 of the above paper was incorrect.The corresponding sentence should read as follows: Oocytes were loaded with the [Ca²⁺]_i indicator fluo-3 (Mintaet al., 1989) by incubation for 45 min at 37°C with 2 µMacetoxymethyl ester of fluo-3 (fluo-3/AM, Sigma) dissolved inB2 medium.     

Human oocyte activation after intracytoplasmic sperm injection

Oocyte activation is a series of events triggered by the fertilizingspermatozoon and necessary for the beginning of the embryonicdevelopment. Calcium plays a pivotal role in this process. Herewe used confocal laser scanning microscopy to examine the changesin the concentration of intra-cellular free calcium ([Ca²⁺])in human oocytes after intracytoplasmic sperm injection (ICSI).The first considerable but short (<2 min) increase in [Ca²⁺]1was detected immediately after the penetration of the micro-injectionneedle into the ooplasm. This rise by itself did not provokeoocyte activation and was also obtained after the injectionof medium without spermatozoa. After a lag period of 4–12h, oocytes that were subsequently activated initiated a secondperiod of [Ca2⁺]1 changes. These changes were sperm-dependentand followed one of two alternative patterns, a non-oscillatoryone and an oscillatory one. The non-oscillatory pattern resembledthe changes described previously during parthenogenetic activationof mammalian oocytes. The oscillatory pattern was similar tothe changes accompanying normal fertilization in different mammalianspecies. It is concluded that the initial [Ca²⁺]1 rise provokedby the ICSI procedure is not responsible for oocyte activation,and that a release of a sperm factor(s) is required to initiatethis process.     

Timing of sperm and oocyte nuclear progression after intracytoplasmic sperm injection

We investigated the time course of human oocyte activation afterintracytoplasmic sperm injection (ICSI) by observing the oocytechromosome configuration at different times after injection.One day old human oocytes were injected with spermatozoa andsubjected to cytogenetic analysis at 2, 3, 4 and 5 h after injection.We found that anaphase is initiated in the vast majority ofthe oocytes between 2 and 3 h after injection, and that by 4–5h after injection most of the oocytes have reached the chromatinmass stage. Two distinguishable stages of sperm nucleus transformationwere observed. The first phase — swelling — wasreached within 2 h after the injection and was independent ofoocyte activation. The second phase — the ‘brush’-likestage or decondensed chromatin stage — was found onlyin activated oocytes. Moreover, this stage was not reached beforethe chromatin mass stage (late telophase) of the oocyte. Thesame proportion of metaphase II oocyte chromosome configurationsand unchanged sperm nuclei was found at any given time afterinjection. We conclude that: (i) ICSI allows users to obtainan almost synchronized population of activated oocytes; (ii)anaphase II is initiated in the majority of oocytes not laterthan 2–3 h after injection and telophase II is reached5 h after injection; and (iii) there are two distinguishablephases of sperm nucleus transformation after ICSI: oocyte activationindependentswelling of the sperm head and oocyte activation-dependent chromatindecondensation which is coupled to the beginning of oocyte chromosomedecondensation.     

High fertilization and implantation rates after intracytoplasmic sperm injection

Previously reported better fertilization rate after intra-cytoplasmicsingle sperm injection (ICSI) than after subzonal inseminationof several spermatozoa was confirmed in a controlled comparisonof the two procedures in 11 patients. Intracytoplasmic sperminjection was carried out in 150 consecutive treatment cyclesof 150 infertile couples, who had failed to have fertilizedoocytes after standard in-vitro fertilization (IVF) proceduresor who were not accepted for IVF because not enough motile spermatozoawere present in the ejaculate. A single spermatozoon was injectedinto the ooplasm of 1409 metaphase II oocytes. Only 117 oocytes(8.3%) were damaged by the procedure and 830 oocytes (64.2%of the successfully injected oocytes) had two distinct pronucleithe morning after the injection procedure. The fertilizationrate was not influenced by semen characteristics. After 24 hof further in-vitro culture, 71.2% of these oocytes developedinto embryos, which were transferred or cryopreserved. Only15 patients did not have embryos replaced. Three-quarters ofthe transfers were triple-embryo transfers. High pregnancy rateswere noticed since 67 pregnancies were achieved, of which 53were clinical, i.e. a total and clinical pregnancy rate of 44.7%and 35.3% per started cycle and 49.6% and 39.2% per embryo transfer.A total of 237 supernumerary embryos were cryopreserved in 71treatment cycles.     

Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection

The association between oocyte morphology and subsequent fertilization rate and embryo quality in intracytoplasmic sperm injection (ICSI) is subject to considerable controversy. This retrospective study was carried out to investigate a possible prognostic value of first polar body morphology with regard to fertilization rate and embryo quality. A total of 70 consecutive ICSI cases was included in this study. The results showed that classification based on first polar body morphology revealed a significant correlation with fertilization rate (P < 0.025) and embryo quality (P < 0.001). Cytoplasmic criteria showed no correlation in this respect. Present data indicate that ICSI of oocytes with intact well-shaped first polar bodies yields higher fertilization rates and higher quality embryos.     

卵子第一极体、卵周隙及胞浆形态与ICSI后受精率和胚胎质量的影响

目的从MII期卵子的第一极体、卵周隙和胞浆形态三方面综合探讨卵子质量对卵母细胞浆内单精子注射(ICSI)后的受精率、卵裂率、优胚获得率的影响.方法对ICSI中去除颗粒细胞后的MII期卵子在光镜下从第一极体、卵周隙和胞浆形态三方面综合评价卵子质量并按形态表现分为五级.且分别比较各级卵子的受精率、卵裂率及优胚率.结果Ⅰ级卵子与其他各级含异常形态的卵子比较,受精率、卵裂率和优质胚胎率,均有显著差异(P＜0.01).Ⅰ级卵子有最高的受精率(91.14%)、卵裂率(96.35%)和优质胚胎率(83.91%).形态异常的卵子的受精率、卵裂率和优质胚胎率均有明显的下降趋势.在取卵数≤15个及hCG注射日每个卵泡的血清E2浓度在732～1098pmol/L组中,可获得高比例的Ⅰ级卵子.结论根据MII期卵子的第一极体、卵周隙及胞浆形态三方面综合因素进行的卵子分级是可预测ICSI后受精率、卵裂率及优胚率.     

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection1

The aim of this study was to determine why oocytes remain unfertilizedor develop three pronuclei after intracytoplasmic sperm injection(ICSI). Unfertilized and abnormally fertilized oocytes werefixed in glutaraldehyde,stained with Hoechst 33342 and examinedby fluorescence microscopy to identify oocyte, sperm and polarbody DNA.One-pronuclear oocytes were considered to be unfertilized.Atotal of 285 unfertilized oocytes were examined (104 ICSI cycles).Overall, 83% of these oocytes were not activated (still at metaphaseII) while 17% had activated and formed a single (female) pronucleus.About 66% of the unfertilized, metaphase II oocytes containeda swollen sperm head, indicating that the oocyte was correctlyinjected but had failed to activate and complete the secondmeiotic division. Premature chromosome condensation of the spermDNA was evident in 6% of these metaphase II oocytes (4% of theunfertilized oocytes). The swollen sperm head was located amongthe oocyte chromosomes in 5%of the metaphase II oocytes. Othercauses of failed fertilization in the metaphase II oocytes werethe failure of sperm head decondensation (11%) and ejectionof the spermatozoon from the oocyte (23%). A similar patternwas observed in one-pronuclear oocytes (52%, swollen sperm head;28%, intact, undecondensed sperm head; 20%, ejection of thespermatozoon), which indicates that asynchronous pronucleardevelopment does not explain the presence of one-pronuclearoocytes. A total of 41 threepronuclear oocytes were examinedand all had a single polar body, which indicates that the retentionof the second polar body leads to the formation of the thirdpronucleus.In conclusion, this study demonstrates that: (i)the major cause of fertilization failure after ICSI is failureof oocyte activation; (ii) ejection of the spermatozoon intothe perivitelline space is not a major cause of fertilizationfailure;and (iii) sperm head decondensation and oocyte activationafter ICSI can occur independently.     

Comparison of pregnancy outcome after intracytoplasmic sperm injection and in-vitro fertilization

The aim of this study was to compare pregnancy characteristics and perinatal outcome of intracytoplasmic sperm injection (ICSI) pregnancies with pregnancies obtained after in-vitro fertilization (IVF). Retrospectively, 145 ICSI pregnancies were matched with 145 IVF pregnancies using the last menstruation data. The main outcome measures were preclinical and clinical abortions, ectopic pregnancies, multiple gestations, prenatal morbidity, prematurity, Caesarean section, birthweight, perinatal mortality and malformations for singletons, twins and triplets. Although patients were significantly younger (P < 0.001) in ICSI (31 years) than in IVF (33 years), their infertility duration (5 years) was similar. The mean number of transferred embryos (2.7 embryos per transfer) was similar in IVF and ICSI. The rates of preclinical (15%) and clinical abortions (11% in ICSI versus 15% in IVF) were not different. Four ectopic pregnancies were observed in the IVF group and none in the ICSI group. In ICSI, two minor malformations were detected and two therapeutic abortions were performed respectively for polymalformations and suspicion of cystic fibrosis. The rate of congenital malformation was 2.8% in ICSI and 2.2% in IVF. In this last group, one therapeutic abortion for malformation of neural tube was performed and two minor malformations were detected. The rate of aborted embryonic sacs before 16 weeks of gestation was not significantly lower in ICSI compared with IVF (13.7% versus 20%). The rate of multiple gestations was similar in both groups (31% in IVF and 35% in ICSI). The number of Caesarean sections was similar in IVF and in ICSI and was twice as frequent for twins versus singletons. The number of singletons born by Caesarean section was 21% after ICSI and 17% after IVF. Mean birthweights and gestational ages at birth for twins were significantly higher (P < 0.05) in ICSI than in IVF (2488 versus 2281 g and 36.5 versus 35.5 weeks). This difference was not observed for singletons. In conclusion, pregnancy characteristics and perinatal outcome after ICSI showed no increase in the number of pathologies in comparison with IVF.      

In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection.

About 4% of all the oocytes denuded prior to intracytoplasmic sperm injection (ICSI) are in metaphase-I (MI). Frequently, these oocytes achieve meiosis after a few hours of in-vitro culture and are available for ICSI on the day of oocyte retrieval. In this retrospective study, the aim was to evaluate the fertilization rate and the developmental capacity of these in-vitro matured MI oocytes. After controlled ovarian stimulation using human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) in 896 ICSI cycles, 1210 MI-to-MII-matured oocytes were injected approximately 4 h after in-vitro culture and 8803 MII oocytes were injected immediately, or later, after denudation. The fertilization rate of in-vitro matured oocytes was significantly lower than that of mature MII oocytes (52.7 and 70.8% respectively, P < 0.00l). Embryo quality was only slightly different as regards the numbers of good quality embryos: 47.4% good quality embryos were obtained in the in-vitro matured oocyte group, whereas 53.2% good quality embryos were obtained in the MII oocyte group (P < 0.05). The same proportions of excellent (5.7 and 7.0%, NS) and fair quality (17.6 and 15.3%, NS) embryos were obtained for in-vitro matured and mature oocytes respectively. Embryos derived from in-vitro matured oocytes were transferred only if they were of better quality or if there were not enough mature oocyte derived embryos available. Fifteen transfers involved only embryos derived from in-vitro matured oocytes: 11 single embryo transfers and four transfers of two embryos, resulting in one singleton pregnancy and the birth of a healthy baby. It may be concluded that in cycles with few MII oocytes it might be worthwhile to inject in-vitro matured MI oocytes in order to increase the number of embryos available for transfer.     

Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection

The world's first deliveries of normal babies after use of flow cytometric separated human sperm cells (MicroSort) for preconception gender selection are reported. Offspring were of the desired female gender in 92.9% of the pregnancies. Most of these pregnancies and births were achieved after simple intrauterine insemination.      

Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.     

Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality

The extent to which the morphology of the oocyte at the light microscopy level is related to the results of intracytoplasmic sperm injection (ICSI) is controversial. In this study, after cumulus removal, oocytes were graded into four groups according to the status of the first polar body, size of the perivitelline space and the presence of cytoplasmic inclusions. Oocyte data from 65 consecutive patients were reviewed. The results showed that, for oocytes without cytoplasmic inclusions, the fertilization rate and embryo development beyond 2-cell stage were significantly lower (P < 0.01) in the oocytes at grade 1-2 (poor) than those in oocytes at grade 3-4 (good). Grade 4 oocytes without inclusions gave the highest proportion (66.7%) of good embryos with grading 1-2 (grade 1 best; P < 0.01). A higher proportion of grade 1-2 oocytes (44.7%; P < 0.05) was obtained from patients older than 35 years. More oocytes containing cytoplasmic inclusions were seen in patients diagnosed as having female factor infertility (24.9%; P < 0.01) and older than 35 years (26.5%; P < 0.05) compared to patients with male factor infertility and younger than 35 years. The fertilization rate and embryo development were not associated with the oestradiol concentration on the day of human chorionic gonadotrophin administration or the total number of oocytes retrieved. The results suggest that human oocyte grading based on the triple factors first polar body, size of perivitelline space and cytoplasmic inclusions is related significantly to fertilization rate and embryo quality after ICSI.      

In-vitro fertilization and intracytoplasmic sperm injection in the treatment of infertility after testicular cancer

Treatment of testicular cancer (TC) may cause infertility due to reduced sperm quality with or without an ejaculation problem. In cases of anejaculation or retrograde ejaculation, spermatozoa can be obtained by transrectal electroejaculation (TE) or testicular sperm extraction (TESE) and used for in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). In this study, 15 out of 17 couples evaluated for infertility after TC, underwent a total of 21 treatment cycles, resulting in 18 embryo transfers. Spermatozoa were obtained by TE in 16 cycles, by masturbation in three cycles and by TESE in one. In one cycle no spermatozoa were found using TESE. Fertilization and cleavage was achieved by IVF in seven cycles and ICSI in 11 cycles; average fertilization rates of 57 and 55% respectively were observed. Twelve clinical pregnancies occurred, of which 11 have been delivered or are ongoing. The ongoing pregnancy rate was 57% per cycle. These results show that infertility after testicular cancer can be treated effectively with IVF and that ICSI even permits treatment of patients who have severe oligozoospermia.      

Randomized comparison of three media used for embryo culture after intracytoplasmic sperm injection

Since the metabolic requirements of fertilization and early embryonic development are very different, we have tested a new culture medium (EllioStep2, Ellios Bio-Media, Paris, France) specially designed for the first cleavages and compared it with two conventional media: BM1 (Ellios Bio-Media, Paris, France) and IVF50 (Scandinavian IVF Science, Gothenburg, Sweden). In order to avoid any interference with fertilization, the test was performed as part of an intracytoplasmic sperm injection (ICSI) study. A total of 416 ICSI attempts were randomly performed using one or other of the media. After sperm injection, oocytes were incubated either in EllioStep2 or in BM1 or in IVF50. The embryo quality, pregnancy and implantation rates, number of frozen embryos were compared in the different media. The percentage of fair embryos (grades 4 and 3) was significantly higher when EllioStep2 was used than when oocytes was cultured in BM1 medium (54 versus 47%; P < 0.01) or in IVF50 (69 versus 61%; P < 0.01). The pregnancy rate per transfer and the implantation rate were not significantly higher with EllioStep2 than with BM1 or IVF50. However, the percentage of embryo freezings per attempt was significantly higher with EllioStep2 than with BM1 (47/105 versus 28/105; P < 0.01). In conclusion, the use of EllioStep2 is associated with an increase in embryo quality permitting a higher number of embryo freezings.