Multinucleation in cleavage stage embryos

BACKGROUND: The aim was to analyse multinucleation in relation to its incidence in time and in the population, and its correlation with clinical variables, with other morphological characteristics and with the implantation rate of cleavage stage embryos. METHODS: Retrospective analysis of 10 388 cleaved embryos from 1395 consecutive IVF/ICSI cycles in 700 patients between January 1, 1999 and June 30, 2002. RESULTS: Multinucleation was observed in 3491 (33.6%) embryos in 1107 cycles (79.4%) of 609 (87%) patients, more frequently on day 2 than on day 3: 2848 (27.4%) versus 1567 (15.1%) [relative risk (RR) = 1.82; 95% confidence interval (CI) = 1.72-1.92]. Its incidence increased with fragmentation: 31.0, 34.4 and 36.5% for fragmentation 相似文献   

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.     

The relationship between chromosomal abnormalities in the human oocyte and fertilization in vitro

The relationship between chromosomal abnormalities in the humanoocyte and fertilization in vitro was investigated by cytogeneticanalysis of an unselected population of oocytes, where failureto achieve fertilization was attributed to dysfunctional spermatozoa.The results demonstrated that 47% of such oocytes were chromosomallyabnormal. These data were used to calculate that the incidenceof chromosomal abnormalities in oocytes that do, and those thatdo not develop pronuclei following insemination in vitro is26.6% and 20.4% respectively. Statistical analysis demonstratedno relationship between chromosomal abnormality in the oocyteand its capacity to achieve fertilization in vitro.     

Cryopreservation of human embryos obtained after gamete intra-Fallopian transfer and/or in-vitro fertilization

During a one-year period 636 excess embryos obtained after in-vitrofertilization and gamete intra-Fallopian transfer combined within-vitro fertilization were cryopreserved using two differentprotocols. For early stage embryos including the pronucleatestage, 1,2-propanediol was used as cryoprotectant (procedureA, adapted from Renard) and for later stage embryos dimethylsulphoxidewas used in protocol B, adapted from Trounson and Mohr. Afterthawing 288 embryos, half of them were of sufficient qualityto be replaced. After cryopreservation, procedure A gave thebest survival in embryos having 2 blastomeres; for later stageembryos best survival was obtained using the dimethylsulphoxideprotocol. Survival after cryopreservation was also clearly relatedto the quality of the embryos prior to freezing. Embryos werereplaced during endocrinologically monitored natural cyclesand were transferred in synchrony between endornetrial and embryonicage. After replacement of 126 embryos in 110 patients, 20 pregnanciesoccurred. So far six healthy children have been born, two patientsaborted and 12 pregnancies are ongoing. In this series no statisticaldifference was observed between the implantation rate of embryoscryopreserved by procedure A or B. Six pregnancies occurredin patients from the oocyte and embryo donation programme. Anadequate cryopreservation programme circumvents the difficultproblem of synchronizing the ovarian cycles of donor and acceptorpatients.     

Chromosome analysis of human oocytes and embryos: does delayed fertilization increase chromosome imbalance?

Thirty per cent of a sample of 120 unfertilized human oocytescarried chromosome abnormalities highly correlated with maternalage (38% in patients >35, as compared with 24% in youngerpatients). Fertilized eggs, when observed 17 h after insemination,showed in 1.6% a single pronudeus suggesting parthenogeitelicactivation. In 92% of the cases two pronuclei were observedand the rate of chromosome anomalies depended on the morphologicalaspect of the embryos. Triploidy was also encountered in 6.4%of the eggs leading to an overall rate of chromosome aberrationsreaching 29.2%. Delayed fertilization drastically increasedthe rate of chromosome anomalIes (87%) as well as the rate ofmosaicism: 30% versus 10.6% in timely fertilized eggs. The highrate of chromosome disorders in early life after in-vitro fertilization(IVF) raises the ethical question of the opportunity of carryingout a genetic control of normality in human embryos at the preimplantationstage.     

Chromosome studies on early human embryos fertilized in vitro

The majority of early spontaneous abortions carry a lethal chromosomalanomaly. While it is recognized that several factors would beresponsible for some IVF failures, it is important to determinethe contribution of chromosomal aberrations to the preimplantationloss of embryos produced in vitro. Chromosome analysis of embryosnot destined for replacement in the uterus could help to elucidatethis phenomenon of early embryonic loss. Fifty-five out of 239embryos fertilized in vitro were successfully karyotyped andamongst these the overall rate of diploidy was 25.5% in thisstudy, which mainly comprised rejected embryos. Embryos withoutcleavage had mostly a chromosomal defect (20/38) and only aminority (9/38) were unfertilized. Numerical abnormalities werefound in a total of 33/46 (71.7%) morphologically normal embryos.In contrast a diploid chromosomal complement was found in only11.1% (1/9) of morphologically abnormal embryos.     

The frequency of chromosome anomalies in human preimplantation embryos after in-vitro fertilization

Previous studies have reported chromosome aberrations in humanpre-embryos after in-vitro fertilization (IVF). Although thereason for these abnormalities is not clear, there is evidencethat they can arise during gametogenesis, fertilization or cleavage.The present study has examined further the incidence of chromosomeabnormalities in human pre-embryos after IVF, using oocytesrecovered from normal volunteer women and from women undergoinginfertility treatment in an embryo-replacement programme. Chromosomepreparations were performed for 75 pre-embryos. Of these 35(47%) gave at least one metaphase in which analysis was possible.The overall incidence of abnormal pre-embryos was 40% (14/35).The absolute frequency of aberrations was 9% for trisomies,3% for polyploidies, 26% for structural anomalies and 3% forhypodiploidies. Five pre-embryos were found to be mosaics, threeof which had each one trisomic metaphase. In five of the pre-embryosmultiple anomalies were found. In 13 of the 14 abnormal pre-embryosthe aberrations were found in only one metaphase. The presentstudy demonstrates that trisomic mosaicism may not be a rareevent in human pre-embryos. Further evidence is provided thatmitotic non-disjunction is important for the production of aberrationsin human pre-embryos     

Pregnancy rate in relation to number of cleaved eggs replaced after in-vitro fertilization in stimulated cycles monitored by serum levels of oestradiol and progesterone as sole index

A simple model for monitoring ovarian stimulation in in-vitrofertilization cycles was designed using daily serum levels of17-oestradiol and progesterone as the only index to determinethe day of ovum retrieval. Human chorionic gonadotrophin wasadministered in the evening 2 days after a level of 2500 pmol/loestradiol in serum was exceeded provided the serum progesteronelevel was > 5 nmol/l. Ovarian stimulation was initiated in128 women scheduled for laparcrscopic oocyte retrieval. Twelvecycles (9%) were cancelled before ovum pick up due to sub-optimalhormone levels in sewn and five for other reasons. In 107 successfullaparclscopies, 616 oocytes (mean 5.8 per lapamcopy) were recovered.The cleavage rate after IVF was 65% in the egg replacement group.In 87 women, a total of 271 cleaved eggs (range 1–6, mean3.1) were replaced. The most important factor for establishinga pregnancy was the number of eggs replaced at the same time.Clinical pregnancies were achieved afkr 30% of replacements,increasing to 50% after replrtce ment of 5–6 eggs. Theongoing/delivered pregnancy rate after the 18th week of gestationwas 69%. It was concluded that the simple monitoring model usedwas consistent with a high pregnancy rate and a low rate ofcancelled cycles.     

A prospective, randomized comparison of ovulation induction using highly purified follicle-stimulating hormone alone and with recombinant human luteinizing hormone in in-vitro fertilization.

The commercial availability of highly purified, s.c. administered urinary follicle stimulating hormone (FSH) preparations for ovarian stimulation marked the beginning of a new era in the treatment of infertility. As these new formulations contain essentially no luteinizing hormone (LH), supplemental LH may be needed for optimal folliculogenesis. It was the aim of this pilot study to compare fertilization rates, embryo morphology, implantation rates and pregnancy outcomes prospectively in two age-matched patient groups: women who received highly purified FSH (FSH-HP) (n = 17), and women who received FSH-HP plus recombinant human LH (rhLH, n = 14) throughout ovarian stimulation. All patients received mid-luteal pituitary down-regulation with s.c. gonadotrophin-releasing hormone agonist (GnRHa) (leuprolide). Mean implantation rates were 26.9 and 11.9% in the FSH-HP only and FSH-HP + rhLH groups respectively. The mean clinical pregnancy/initiated cycle rate was 64.7 and 35.7% for the FSH-HP only and FSH-HP + rhLH patients respectively. FSH-HP patients and FSH-HP + rhLH patients achieved clinical pregnancy/transfer rates of 68.8 and 45.5% respectively. One patient in the FSH-HP + rhLH group had a spontaneous abortion; no pregnancy losses occurred in the FSH-HP only group. There were more cancellations for poor ovarian response among FSH-HP + rhLH patients (n = 3) than among FSH-HP patients (n = 1). The trend toward better pregnancy outcomes among patients who received FSH-HP without supplemental rhLH did not reach statistical significance. It is postulated that appropriate endogenous LH concentrations exist despite luteal GnRHa pituitary suppression, thereby obviating the need for supplemental LH administration.     

Fertilization and early embryology: Detection of fertilization in embryos with accelerated cleavage by fluorescent in-situ hybridization (FISH)

Embryos from a couple undergoing routine in-vitro fertilizationfor unexplained infertility had shown cleavage by day 1 in twoconsecutive cycls. The response of the women to fertility drugshad been normal, and there were no known sperm abnormalities.In a subsequent cycle, accelerated cleavage occurred again andwe used a modified method of spreading whole embryos and dualfluorescent in-situ hybridization (FISH) with directly labelledprobes for chromosomes X and Y to determine if fertilizationhad occurred in these embryos. Nine oocytes were collected,five of which had cleaved when examined for pronuclei earlyon day 1 following late insemination. Pronuclei were observedin one of the remaining oocytes, but in this case, three werepresent. On day 2, all of the oocytes/embryos had cleaved andtwo were transferred to the patient. The remaining seven werespread for FISH analysis but nuclei were only obtained fromfive. In three, a Y signal was detected, indicating that fertilizationhad occurred. In all five embryos, a wide range of X chromosomesignals were observed. These data suggest that the embryos hadundergone abnormal fertilization and accelerated cleavage.     

Dexamethasone during ovulation induction for in-vitro fertilization: a pilot study

The purpose of the present study was to determine whether adrenalandrogen suppression with dexamethasone (DEX) during ovulationinduction improves the outcome of in-vitro fertilization (IVF)cycles. A total of 25 patients with serum dehydroepiandrosteronesulphate (DHEAS) concentrations>2.5 µg/ml were randomizedto receive either 0.5 mg DEX daily or placebo during ovulationinduction with leuprolide acetate down-regulation plus humanmenopausal gonadotrophins (HMG). Nine patients undergoing asubsequent IVF cycle were crossed over to the other treatmentgroup. Ovarian responsiveness and IVF outcome variables analysedincluded number of follicles>12 mm in diameter, serum oestradiolconcentrations on the day of human chorionic gonadotrophin (HCG)administration, number of ampoules of HMG administered, numberof oocytes retrieved, percentage of oocytes fertilized, numberof embryos transferred, implantation rate and numbers of clinicalpregnancies and live birth pregnancies. The 31 randomized IVFcycles revealed a trend towards a higher implantation rate forthe placebo-treated group compared to the DEX-treated group(24 versus 10%P=0.07). The remainder of the IVF cycle variablesrevealed no statistically significant differences. In conclusion,the suppression of adrenal androgens with DEX in women withDHEAS concentrations >2.5 µg/ml appears to have nobeneficial effects on ovarian responsiveness or clinical orlive birth pregnancy rates.     

Early embryo cleavage is a strong indicator of embryo quality in human IVF.

BACKGROUND: In order to decrease multiple birth rates without decreasing birth rates overall, it is important to increase the capability of selecting the most optimal embryos for transfer. It has been shown that human embryos which cleave early, i.e. complete the first mitotic division within 25-27 h post insemination, provide higher pregnancy and implantation rates. METHODS AND RESULTS: In this prospective study, an evaluation of 10 798 scored embryos showed that early cleavage resulted in a significantly higher proportion of good quality embryos compared with late cleavage (62.5 versus 33.4%, P < 0.0001). When examining both day 2 and day 3 transfers together, early-cleaving embryos (306 transfers) gave rise to significantly higher rates of pregnancy/transfer (40.5 versus 31.3%, P = 0.0049), implantation (28.0 versus 19.5%, P = 0.0001) and birth/ongoing pregnancy (34.3 versus 24.0%, P = 0.0009) than did late-cleaving embryos (521 transfers). A stepwise logistic regression of all data showed that the total number of good quality embryos and female age were independent predictors of both pregnancies and birth. For intracytoplasmic sperm injection (ICSI) embryos, early cleavage was found to be an independent predictor of birth. CONCLUSIONS: Early embryo cleavage is a strong biological indicator of embryo potential, and may be used as an additional embryo selection factor for ICSI embryos.     

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis

Normally fertilized human embryos were biopsied at cleavagestages on the third day after in-vitro fertilization (IVF).One or two blastomeres at the 8-cell stage were removed andco-cultured with the biopsied embryos. Embryos and blastomereswere assessed daily for morphological development until day6, when the number of cells were counted by labelling the nuclei.In all, 53% of the biopsied embryos (25 out of 47) reached theblastocyst stage between day 5 and 6 and the proportion wasthe same irrespective of the number of cells removed. Therewas no significant difference between biopsied embryos fromwhich one or two blastomeres respectively had been removed withregard to total cell numbers at the blastocyst stage (56.2 ±3.0 and 64.7 ± 5.5), number of trophectoderm (45.4 ±3.5 and 44.0 ± 5.7) and inner cell mass cells (14.0 ±1.2 and 16.6 ± 1.8). Overall, 72% of the isolated blastomeresdivided at least once over 3 days in culture and 50% dividedmore than once. The mean overall cell number after 3 days inculture was 3.7 ± 0.48 per blastomere (range 1–8cells) if one cell was removed and 6.9 ± 1.0 if two cellswere removed. If the undivided blastomeres are excluded, themean cell number was 4.8 ± 0.51 and 8.3 ± 1.0respectively. Over this period, 55% of the blastomeres cavitated.Of the blastomeres taken from embryos that developed to theblastocyst stage, 92% divided and 76% cavitated. In those fromarrested embryos, 50% divided (P < 0.002) and 32% cavitated(P < 0.003). From the first group 8% of blastomeres and fromthe second group 41% of blastomeres neither divided nor cavitated(P < 0.008). We conclude that as a more consistent alternativeto blastocyst biopsy, cryopreserving biopsied cleavage stageembryos and culturing blastomeres would increase the numberof cells available for genetic analysis. This could facilitatepreimplantation diagnosis of inherited disease by improvingreliability and possibly allowing combined detection of chromosomaland single cell defects. Further studies will investigate thegenetics of proliferated blastomeres.     

Chromosome investigations in early life. II. Human preimplantation embryos

Cytogenetic analysis of 68 human embryos at the 2-to 8-cellstage was performed according to Tarkowski's technique. Sixteenper cent of diploid embryos showed abnormalities, essentiallydiploid/haploid or triploid/haploid mosaicism. Considering theaspect of the embryos, 11% of healthy looking and 19% of fragmentedembryos were chromosomally abnormal without, however, any statisticalsignificance in this small series. Only 46.7% of the tripronucleatefertilized eggs showed a triploid chromosome complement. In20% of the cases, however, diploid metaphases were found, andin the last 30% a triploid/diploid mosaicism. One per cent ofthe oocytes displayed a single pronucleus, and the resultingembryos contained haploid sets of chromosomes suggesting a parthogeneticactivation. The overall rate of chromosome abnormalities, including16/ of abnormal diploid eggs, 6/ of polyploid and 1/ of haploidembryos, thus reaches 23/ in this series.     

A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.     

Cytogenetic study of human oocytes uncleaved after in-vitro fertilization

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.     

A double-blind cross-over controlled study to evaluate the effect of human biosynthetic growth hormone on ovarian stimulation in previous poor responders to in-vitro fertilization

The effect of exogenous human biosynthetic growth hormone (HGH;12 IU/day; Norditropin, Novo-Nordisk) on the response to ovarianstimulation using a buserelin/human menopausal gonadotrophin(HMG) regimen was assessed in women who had previously showna ‘poor response’ in spite of increasing doses ofHMG. Forty patients were recruited into a prospective double-blindplacebo-controlled study. The serum follicle stimulating hormone(FSH) on day 2–5 of a menstrual cycle (< 10 IU/I) wasused to exclude any peri-menopausal candidates. The urinary24 h GH secretion was normal in all patients. Thirty-three patientscompleted the study with 21 patients having human chorionicgonadotrophin (HCG) in both arms, thus providing a completeset of placebo control data. Of these 21 patients, the administrationof HGH compared to the placebo cycle resulted in increased serumconcentrations of fasting insulin on the 8th (median 3.9 versus5.8 mU/I; P < 0.0005) and13th (median 4.4 versus 5.8 mU/I;P < 0.05) day of HMG in those cycles receiving HGH. After8 days of co-treatment with HGH the number of cohort follicles(14–16.9 mm) was significantly increased, but this changewas not sustained on the day of HCG administration. No statisticaldifference in the serum oestradiol on the 8th day of HMG orday of HCG, length of the follicular phase, total dose of HMGused, or the number of oocytes collected was seen between theplacebo or HGH cycles. This study demonstrates that HGH doesnot improve the ovarian response to ovulation induction in previouspoor responders.     

The effect of preinsemination interval upon fertilization of human oocytes in vitro

A correlation between oocyte maturity, duration of preinseminationinterval (PII) and fertilization rate in vitro has been suggested.Therefore, delayed insemination is being widely practised inmany IVF centres. The purpose of this study was to investigateprospectively the effect of PII upon the fertilization ratein vitro. A total of 1474 oocytes were inseminated followingincubation for between 30 and 540 min. The duration of the PIIwas determined randomly. It was found that neither fertilizationrate (on average 46.9%) nor pregnancy rate (20.4%, calculatedper embryo transfer) were affected by PII for any given degreeof oocyte-cumulus maturity. The ability to inseminate oocytesat any time (within 9 h following egg collection) to the convenienceof the biologist further simplifies the technology of IVF.     

Preliminary experience of the use of a gonadotrophin-releasing hormone antagonist in ovulation induction/in-vitro fertilization prior to cancer treatment.

Therapeutic regimens for the treatment of malignant disease may compromise future fertility. One approach to circumvent this is the cryopreservation of embryos created before treatment for the malignancy. Conventional regimens using gonadotrophin-releasing hormone (GnRH) agonists are time consuming, requiring pituitary down-regulation before gonadotrophin administration, thus the duration of treatment is approximately 20-30 days. GnRH antagonists, however, do not cause an initial stimulation of gonadotrophin secretion and can thus be administered during the later stages of follicular maturation to prevent premature luteinization and ovulation. The duration of ovulation induction/in-vitro fertilization (IVF) treatment is thus reduced. In this study, case histories are reported of six women with newly diagnosed malignancies who requested ovulation induction/IVF prior to chemotherapy or surgery in which we have used the GnRH antagonist Cetrorelix. Gonadotrophin administration was started in the early follicular phase, and Cetrorelix (0.25 mg s.c. daily) was added from day 6 of treatment. Subsequent to human chorionic gonadotrophin (HCG) administration oocytes were recovered and successful fertilization and embryo cryopreservation was achieved in all cases. The median duration of treatment was 12 days (range 8-13, including induction of luteolysis in two patients). These results illustrate the potential use and advantages of a GnRH antagonist in ovulation induction/IVF when the need for immediate initiation of treatment and its duration are critical factors.