Premature chromosome condensation after in-vitro fertilization

During an in-vitro fertilization programme, 320 inseminatedoocytes neither formation of pronucle nor cell cleavage werestudied Cytogentically. Fourteen of 17 oocytes exhibiting noextruction of polar were characterized by an approximately diploidset of metaphase II chromosomes with four of these oocytes alsoshowing an additional set of prematurely condensed sperm chromosomesof the G₁ phase (G₁ -PCC). These chromosomes were single chomatids.Among 211 occytes characterized by polar body extrusion, thesame type of chromosomes were found in 22, along with metaphaseII chromosomes in the haploid range. This phenomenon can beexplained by the permanent arrest of the oocytes at metaphaseII after sperm penetration, which allows the presence of thepermanent arrest of the oocytes at metaphase II after spermpenetration, which allows the presence of cytoplasmic chromosomecondensing factors to remain, leading to the induction of PCCin the sperm. In these cases, PCC resulted either from distinctpronuclear asnchtrony or interchromosomal asynchrony withinthe chromosome set.     

Fertilization and early embryology: Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection

A cytogenetic-cytological study was performed on unfertilizedhuman oocytes (first polar body visible) after intracytoplasmicsperm injection (ICSI) with respect to the rate of prematurelycondensed sperm chromosomes (G₁-PCC). Out of 163 prepared oocytesderived from 41 ICSI cycles, 133 (-82%) could be analysed successfully.A total of 60 oocytes (45.1%) showed metaphase II chromosomesin the haploid range along with an intact sperm head and 27oocytes (20.3%) were missing the sperm head, but two of themshowed an approximately diploid set of chromosomes; 38 oocytes(28.6%) exhibited the maternal metaphase II chromosomes as wellas G₁-PCC of the sperm nucleus showing a remarkable variationin the degree of condensation. Ten ICSI cycles (each followedby an embryo transfer) were characterized each by 2–3oocytes demonstrating G₁-PCC. It is concluded that the maincause of failed fertilization after ICSI is the failure of oocyteactivation. When the sperm nucleus is able to act with the chromosomecondensing factors and the oocyte does not become activated,this will lead to the induction of PCC. Absence of the spermhead might be due to injection or ejection of the spermatozoonin the perivitelline space except for two cases in which fertilizationmight have occurred. Finally, the observation of both a singlechromatin region (n = 6) or two chromatin regions (n = 2) indicatedoocyte activation which, however, was followed by developmentalarrest.     

Abnormalities of sperm chromosome condensation in the cytoplasm of immature human oocytes

This study analysed data from 27 couples in an IVF-ET programme. The maternal age range was 28-43 years. Statistical analyses on 182 oocytes showed no maternal age effect on the number of oocytes, their stage of maturation or their fertilization rate. There was also no effect of age of either partner or of seminal parameters on the fertilization rate. In contrast, occurrence of diploid oocytes was confined to three of the older women. The proportion of failures of fertilization was significantly higher in immature oocytes. These failures, which included 18 uncleaved, multipronuclear or fragmented zygotes, were related to disturbances of oocyte maturation. Four (out of five) oocytes re-inseminated with fresh semen produced polyspermy. One zygote showed marked asynchrony in the development of the two pronuclei. In eight zygotes the paternal complements had an allocyclic pattern of chromosome condensation between and within chromosomes or chromosome regions. In two other zygotes the paternal complement showed one chromosome prematurely condensed. This single-chromatid chromosome would be lost in the following cleavage division, suggesting that aneuploidy due to 'anaphase lag' is not a rare event during embryo cleavage.     

Mouse spermatid nuclei can support full term development after premature chromosome condensation within mature oocytes.

The nucleus of round spermatids, the earliest haploid male germ cells, can participate in the formation of normal zygotes when incorporated into activated oocytes. In this study, we injected mouse round spermatids into homologous mature oocytes that were kept arrested at metaphase II to induce premature chromosome condensation (PCC) of the spermatid nuclei. After full condensation of the spermatid chromosomes, the oocytes were activated by Sr2+-containing medium, into which cytochalasin B was added to prevent extrusion of the segregated female and male chromosomes as polar bodies. Out of 142 oocytes examined, 104 (73%) formed two male (pseudo)pronuclei and two female pronuclei. To restore the diploid state of these zygotes, one of the female pronuclei was removed. When cultured in vitro for 72 hours, all (n = 37) of the constructed embryos developed to the morula/blastocyst stage. When 2-cell embryos and morulae/blastocysts were transferred into pseudopregnant females, 14 (13/96) and 24% (9/37), respectively, developed into term offspring. This study indicates that the spermatid chromosomes, which had undergone PCC, moved safely to opposite poles after oocyte activation. Since round spermatids contain no (in the mouse) or little (in patients with spermatogenic failure) oocyte-activating factor, this method may be used to rescue oocytes that fail to be activated at the time of spermatid injection.     

High rate of premature chromosome condensation in human oocytes following microinjection with round-headed sperm: case report

A young couple proceeded to three ICSI treatment cycles becauseof male infertility. The semen samples varied between 10 x 10⁶and 36 x 10⁶/ml, 38 and 51% progressive motility but 0% normalmorphology. Different types of sperm heads, mostly round-headedwith varying spherical appearance (86%) were presented besideacephalic sperm (pinheads; 12%), both one- or two-tailed andthe former also without a tail. Very few sperm (2%) exhibitedslightly oval-shaped heads. Electron microscopy revealed theabsence of the acrosome combined with disturbance of the chromatincondensation among the round-headed sperm. In all three cycles,the fertilization rate using the round-headed sperm fractionwas very low with the best result of 2/18 (11%) two-pronucleateoocytes and one one-pronucleate oocyte obtained in the secondICSI cycle. The three oocytes cleaved and were transferred inthe 3–4-cell stage without achieving a pregnancy. Of the29 unfertilized and prepared oocytes from the last two cycles,27 were informative and revealed the maternal metaphase II chromosomesin the haploid range and a high rate (85%) of premature chromosomecondensation (PCC) of the sperm nucleus with remarkable variationin the degree of condensation. Thus, it appears that nearlyall round-headed sperm from this patient were incapable of oocyteactivation after ICSI, which could be due to non-release (orabsence) of an activating factor. As a consequence, PCC wasinduced in the sperm nuclei by the chromosome condensing factorswhich were still active in the oopasm of the arrested oocytes.     

Pregnancy: Premature luteinization as detected by elevated serum progesterone is associated with a higher pregnancy rate in donor oocyte in-vitro fertilization

Premature luteinization has been reported to be associated withdecreased pregnancy rates in patients undergoing in-vitro fertilization.However, the detrimental effect created by a pre-aspirationrise in progesterone is difficult to assess since ovarian stimulationaffects both oocyte quality and endometrial receptivity. Therefore,the relationship between premature luteinization and pregnancyrates remains uncertain. To achieve improved control for confoundingvariables, we studied premature luteinization in ovum donorsof proven fertility. A total of 114 consecutive ovum donationcycles using pituitary suppression with a gonadotrophin-releasinghormone agonist followed by gonadotrophin stimulation were examined.Serum progesterone concentration on the day of administrationof human chorionic gonadotrophin (HCG) was > 1.2 ng/ml in29% of patients. Patients were divided into two groups basedon this value. There was a significant increase in clinicalpregnancy rates per embryo transfer in the group with higherprogesterone concentrations (53 versus 25%, P = 0.012), as wellas significantly more oocytes obtained at aspiration (19.6 ±10.4 versus 13.3 ± 5.4, P < 0.001), and significantlyhigher peak serum oestradiol values (3903 ± 1787 versus2453 ± 1232 pg/ml, P < 0.001). There were no significantdifferences between groups due to age, degree of stimulationor the number of embryos transferred. We conclude that prematureluteinization as based on elevated serum progesterone concentrationis a common occurrence in oocyte donors, reflects healthy folliculardevelopment, and is associated with increased pregnancy rates.     

Developmental and functional evidence of nuclear immaturity in prepubertal oocytes

BACKGROUND: The nuclear compartment has been proposed as responsible for the developmental arrest of prepubertal mouse oocytes while the studies on prepubertal sheep and cow oocyte model suggested the cytoplasm immaturity accounts for this failure. METHODS: The apparent disagreement on the causes of developmental defects between these two species prompted us to study: (i) follicular and oocyte growth allometry in lambs, (ii) oocyte compartment (nucleus versus cytoplasm) responsible for developmental failure by nucleus exchange between lamb and adult sheep oocytes, (iii) nucleolar features of prepubertal oocytes by ultrastructural observation and (iv) in vivo developmental survey of prepubertally derived embryos. RESULTS: The oocyte growth inside the follicle is asynchronous during prepuberty. The nuclear transfer revealed that the lamb nucleus was responsible for developmental failure. Immature fibrillogranular structure of the nucleolus has been revealed in small lamb oocytes and also in a few adult-size lamb oocytes. Studies in vivo revealed a high occurrence of developmental arrest of prepubertal derived fetuses, which we have attributed to the low genome-wide methylation detected in prepubertal oocytes. CONCLUSIONS: Our studies have indicated incomplete nuclear maturation of prepubertal gamete. The implication of this finding suggests caution when the strategy of rescue of prepubertal oocytes for assisted fertilization is considered such as in the case of therapeutic treatment which precludes the maintenance of fertility of sexually immature patients.     

Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles

BACKGROUND: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. METHODS: Two cryopreservation procedures were used. Method 1 involved use of 1.5 mol/l propanediol (PrOH)-0.1 mol/l sucrose with medium containing sodium (Na) as cryoprotectant medium, seeding at -7 degrees C, and stepwise dilution of cryoprotectant post-thaw. Method 2 used Na-depleted media with 1.5 mol/l PrOH-0.2 mol/l sucrose for freezing, seeding at -6 degrees C, and use of high sucrose (0.5 and 0.2 mol/l) for cryoprotectant removal. RESULTS: The first method was used in seven patients, and gave poor (12.3%) survival results and no pregnancies. The second method was used in 15 patients (16 cycles), and yielded good survival and fertilization rates (74.4 and 59% respectively), with four pregnancies and five healthy infants born to 11 women receiving an embryo transfer. CONCLUSIONS: Using Na-depleted media along with other alterations in freezing and thawing procedures, human oocyte cryopreservation can provide excellent survival and pregnancy rates.     

Acute ureteral obstruction following transvaginal oocyte retrieval for IVF.

Transvaginal, ultrasound-guided oocyte retrieval has become the gold standard for IVF therapy. Despite a low reported complication rate, here a case is reported of acute ureteral obstruction following seemingly uncomplicated oocyte retrieval. Prompt diagnosis and ureteral stenting led to rapid patient recovery with no long-term urinary tract sequelae. Ureteral injury needs to be included in the differential diagnosis of a patient presenting with pelvic/abdominal pain following oocyte retrieval.     

Fertilization and early embryology: Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation

All oocytes from a patient who had undergone four unsuccessfulin-vitro fertilization attempts showed neither a polar bodynor pronuclei when examined for fertilization. In 19 inseminatedoocytes that were spread for karyotypic analysis, one haploidset of metaphase II chromosomes and a remarkable condensed structurewere found. Hormonal and morphological criteria implied thatthe oocytes had been mature at the time of retrieval. Sincenon-inseminated oocytes contained only one set of metaphaseII chromosomes, the condensed structure appeared to representthe sperm chromatin in the state of premature chromosome condensationdue to a block in oocyte maturation. Since the first and secondpolar body, as well as their chromatin, were undetectable inall the patient's oocytes, a rapid maturation to metaphase IIbefore retrieval and prolonged arrest in this state before fertilization,accompanied by degeneration of the first polar body, appearto be responsible for the condition. In accordance with thisnotion, degenerate spindles (typical of post-ovulatory agedoocytes) and separating chromosomes (probably representing presegregatingchromatids) were observed by antitubulin immunofluorescence.     

The role of oocyte donation in women who are unsuccessful with in-vitro fertilization treatment.

Oocyte donation improves the chances of becoming pregnant in some women who are unsuccessful with in-vitro fertilization (IVF) treatment. A total of 119 IVF cycles achieved a pregnancy rate per cycle of 2.5% whereas the same women, when treated with 45 cycles of oocyte donation, achieved a 24.5% pregnancy rate per cycle. To ascertain which women may be helped by oocyte donation, IVF data were analysed according to the outcome of oocyte donation. There was a difference in the number of previous natural conceptions and live births, and in the IVF fertilization rate. There was no difference in the age of the women and the numbers of oocytes collected per cycle of IVF. New criteria are therefore suggested for recommending oocyte donation to women who have previously failed to become pregnant with IVF treatment.     

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.     

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 survey of anonymous oocyte donors: demographics

This is a questionnaire based study of 501 women enquiring aboutanonymous oocyte donation at a private in-vitro fertilization(IVF) unit, investigating the demographic characteristics andlogistic issues involved in ovum donation. The 501 women weremade up of 356 women who did not donate (‘non-donors’).Although there was a majority of housewives among the enquirers,women in full-time employment were the majority of actual donors.Logistic factors such as the travel and time commitment involvedwere major reasons for non-donation as well as concerns aboutcomplications. There was a paucity of ethnic donors. Recruitmentstrategies must focus on retaining potential donors and ensuringa higher proportion become actual donors. These strategies mustaddress the logistic difficulties associated with non-donationincluding transport problems and social commitments by assistingwith childcare provision and travel. Improving donor educationand the access to more personal and non-threatening informationwere other areas that needed attention which were highlightedin the survey.     

Granulosa cell metabolism and the assessment of oocyte quality in IVF

The progesterone production of the granulosa cells of the cumulus– oocyte complex correlates very well with the cleavagepotential of embryos in an IVF system. The method is simpleand can be easily performed by any laboratory associated withIVF. Furthermore, high intratubal progesterone levels in theimmediate post-ovulatory period are probably important in prolongingthe intra-ampullary residence of the oocyte or embryo untilthe uterine endometnum is optimal for implantation.     

G-banding and chromosome condensation in the ant,Tapinoma nigerrimum

Well-defined G-bands were obtained on metaphase chromosomes fromTapinoma nigerrimum using trypsin and warm 2×SSC in sequence. The G-banded pattern allowed the identification of all chromosomes. Evidence for asynchronous condensation of the chromosomes of this species is provided. Different banding patterns were obtained when metaphase chromosomes were stained with DA/DAPI alone and with DA/DAPI after a standard G-banding procedure. The G-banding phenomenon is discussed using the result obtained.accepted for publication by H. C. Macgregor     

Ultrasound-guided hydrosalpinx aspiration during oocyte collection improves pregnancy outcome in IVF: a randomized controlled trial

BACKGROUND: Hydrosalpinges have adverse effects on IVF outcomes. Salpingectomy is effective in improving outcomes, but it is not always practical or safe. Ultrasound-guided aspiration of hydrosalpinges at oocyte collection is an option for those who develop hydrosalpinges during controlled ovarian stimulation; however, there is no randomized evidence to show whether this practice is effective. METHODS: Between October 1999 and June 2003, consenting women of age 相似文献   

Auricular electro-acupuncture as an additional perioperative analgesic method during oocyte aspiration in IVF treatment

BACKGROUND: The aim of this study was to compare the pain-relieving effect and the subjective well-being between auricular electro-acupuncture (EA) analgesia, auricular acupuncture (A) and conventional analgesia with remifentanil (CO). METHODS: A total of 94 women undergoing IVF were randomized to auricular acupuncture with (EA, n = 32) or without (A, n = 32) continuous 1 Hz auricular stimulation (using a battery-powered miniaturized stimulator, P-Stim) or with adhesive tapes instead of needles and no electrical stimulation (control group, CO, n = 30) at the auricular acupuncture points 29, 55 and 57. All patients received patient-controlled analgesia (PCA) with remifentanil. Pain intensity and psychological well-being were assessed by means of visual analogue scales (VAS); tiredness, nausea and vomiting and analgesic drug consumption were documented. RESULTS: Pain relief and subjective well-being were significantly greater in group EA during and after the procedure as compared with groups A and CO (P < 0.001). The patients were significantly more tired in group CO than in groups A and EA (P < 0.001). Consumption of the opioid remifentanil was significantly lower in group EA, comparable nausea (P < 0.001). CONCLUSION: Auricular EA significantly reduces pain intensity and analgesic consumption of the opioid remifentanil during oocyte aspiration in IVF treatment.     

Significance of cumulus oophorus in in-vitro fertilization and oocyte viability and fertility

Fertilization and cleavage rates of human cumulus-intact oocytes incubated in vitro for 36-48 h with normal spermatozoa tended to be higher than those which were cumulus-denuded (73 versus 68%; 68 versus 56%, respectively); however, the difference was not significant. Nor were these differences significant when using sperm samples of various qualities (normozoospermic samples: 75 versus 70% fertilized oocytes; asthenozoospermic: 66 versus 64%; oligozoospermic: 64 versus 56%; oligoasthenozoospermic: 35 versus 33%). The beneficial effect of the human cumulus oophorus on the binding of human spermatozoa to denuded hamster oocytes and on head decondensation of human spermatozoa observed after 2 h of incubation (9.3 versus 7.0 bound spermatozoa per oocyte, P less than 0.05; 0.5 versus 0.3 decondensed sperm heads per oocyte, P less than 0.02) disappeared after 6 h. A protective effect of the cumulus oophorus on hamster oocytes preincubated in medium containing 50% human preovulatory follicular fluid was observed in the sperm penetration assay (fertilization rate of cumulus-intact: cumulus-denuded oocytes, 26 versus 13%, P less than 0.05) and confirmed using fluorescein diacetate stain (cumulus-intact oocytes: 86 versus 100% vitality, non-significant; cumulus-denuded oocytes: 64 versus 100%, P less than 0.01). These data suggest the accelerating effect of the human cumulus oophorus on fertilization in its early stages. Furthermore, the cumulus plays an important part in protecting the oocyte against adverse environmental influences.     

Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells

Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.