Cytogenetic analysis of unfertilized human oocytes

Cytogenetic studies were carried out on 180 oocytes that appeared unfertilized after in-vitro fertilization. The majority of the 135 that were informative had grossly haploid second meiotic metaphases, two were grossly diploid, and five had a variety of different abnormalities. Twenty-one oocytes were abnormally fertilized and included prematurely condensed sperm chromosomes. The frequency of this phenomenon varied according to the stimulation protocol, those oocytes maturing longer in vivo showing less propensity to abnormal fertilizations. Thirteen per cent of the analysable haploid metaphases were hyperhaploid but none contained extra whole chromosomes. The extra components were a single chromatid (one case), or two single chromatids replacing a whole chromosome (four cases). The data suggest that the chromatids arose as a result of premature centromere division at meiosis I, and that this may be a major mechanism for trisomy formation rather than non-disjunction of whole bivalents at meiosis I, as generally believed.     

Cytogenetic analysis of human oocytes from fertile women.

Several reports have shown a relatively high incidence of chromosome anomalies in human inseminated-unfertilized oocytes from infertile women. In the present study, cytogenetic analysis was attempted in 73 human oocytes from 17 fertile women in order to establish the incidence of chromosome anomalies in the fertile population and to compare this with the incidence in inseminated-unfertilized oocytes from infertile women. Of 56 oocytes that could be analysed, 42 oocytes were haploid, 12 were hypohaploid and two exhibited fragmented chromosomes. The low rate of chromosome anomalies (3.6%) found in this population suggests that there is natural selection at fertilization against diploid oocytes and oocytes with fragmented chromosomes. This result also questions the high incidence of chromosome anomalies found in previous reports using inseminated-unfertilized oocytes.     

The frequency of chromosome anomalies in human unfertilized oocytes and uncleaved zygotes after insemination in vitro

Data on first trimester human abortions have shown chromosomeaberrations in 50% of the cases, most of which were trisomies.More than 80% of these trisomies have been attributed to anerror in the oocyte. In this study we investigated the frequencyof chromosome anomalies in 44 human unfertilized oocytes and13 uncleaved zygotes after insemination in vitro. The oocyteswere aspirated from preovulatory follicles in porous volunteerdonors and patients participating in an embryo replacement programme.Thirty cases could be analysed, 18 of which (60%) carried achromosome abnormality. There were two hyperhaploidies (6.7%),four hypohaploldles (13.3%), three polyploidies (10%), six withstructural aberrations (20%) and five diploidies (16.7%). Theincidence of these abnormalities, except for the structuralanomalies, was similar to that reported previously. The higherfrequency of structural aberrations in the present study maybe related to delayed cytogenetlc analysis for up to 48 h afterInsemination.     

Cytogenetic analysis of human oocytes and embryos in an in-vitro fertilization programme.

Oocytes (unfertilized and preovulatory) and embryos (normal and polypronuclear), which were donated to research by patients undergoing procedures of assisted reproductive treatment, were analysed for cytogenetic abnormalities. A total of 362 oocytes and embryos were analysed. The unfertilized oocytes with readable metaphases (53.4%) gave 25.2% chromosomal abnormality with diploidy being the main aberration observed. A high incidence of premature chromosome condensation (PCC) was observed and the incidence of PCC in oocytes exposed to colcemid was significantly higher (14/62, 22.6%) than in those not exposed to this treatment (3/41, 7.3%, P less than 0.05). When chromosomal anomalies and PCC in the unfertilized oocytes were correlated to various patient criteria such as stimulation regimen, number of human menopausal gonadotrophin ampoules, peak oestradiol levels, age of patient and number of previous attempts, none of the criteria tested had any significant relationship to the incidence of chromosomal abnormality. However a significant increase in the incidence of PCC was noted in the gonadotrophin-releasing hormone (GnRH) 'flare' group (6/15, 40.0%) compared to the GnRH 'down-regulation' group (11/88, 12.5%). The incidence of chromosomal abnormalities among preovulatory oocytes was 16.7% and diploidy was the only abnormality noted. For embryos arising from two-pronuclear oocytes, the chromosomal constitution related mainly to embryo quality. The rate of chromosomal abnormality for apparently good quality embryos was 23.5% and for poor or fragmented embryos 83.3%. The majority (77.3%) of the readable metaphase plates for polypronuclear 1-cell and cleaved embryos showed grossly abnormal chromosome complements but 19% of the cleaved embryos contained sets of normal diploid chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization

At 16–18 h after insemination, 5.5% of the inseminatedoocytes displayed only one pronucleus. The incidence of single-pronucleatedoocytes was not correlated with the age of the patient. Cytogeneticanalysis of 41 embryos derived from single-pronucleated oocytesrevealed a haploid chromosome complement in 12.2%, a diploidchromosome complement in 80.5% and a triploid set in 7.3% ofthe embryos. In one diploid metaphase, a Y chromosome couldbe clearly demonstrated. A total of 312 single-pronucleatedoocytes were evaluated twice (at 16–18 h and at 20–24h after insemination). In 25% of the single-pronucleated oocytesa second pronucleus was observed 4–6 h later, suggestingasynchronous or delayed pronuclear formation. The cleavage ofthese embryos was similar to the cleavage of embryos with twopronuclei at the first evaluation. Parthenogenetic activationand asynchronous pronuclei may both be mechanisms leading tothe morphological observation of a single pronucleus. In clinicalpractice, the second repeat observation of single-pronucleatedoocytes became part of the standard procedure.     

Endocrine changes in women conceiving during treatment with an LHRH agonist

We report on eight patients who conceived during pituitary desensitization with buserelin in the luteal phase of the menstrual cycle. Pregnancy was diagnosed between day 12 and 21 of buserelin administration. Analysis of serum luteinizing hormone on day 12 showed that pituitary desensitization occurred in conjunction with increasing production of ovarian steroid hormones. Serum concentrations of human chorionic gonadotrophin (HCG) were less than 10 IU/l on day 1 of buserelin administration for seven of the eight patients. The serum concentration of HCG on day 12 showed a median value of 722 IU/l (range 14.6-798 IU/l). Five of the eight patients were given HCG support (10,000 IU) following the diagnosis of pregnancy--three of these patients have ongoing pregnancies and the remaining two had blighted ova on scan. Of the remaining three patients, one had a singleton pregnancy which miscarried at 9 weeks, one had a blighted ovum on scan and bled per vagina shortly after this, and one bled per vagina prior to a scan being carried out. Our results show that pregnancy can occur during pituitary desensitization with buserelin, despite patients being counselled not to have unprotected intercourse in the cycle during which administration commences. An HCG assay on day 1 of buserelin administration is not helpful. Pregnancy should be suspected when ovarian steroid production persists despite complete pituitary down-regulation.     

Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome

This case report describes the birth of a healthy infant after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from an anovulatory woman with polycystic ovarian syndrome (PCOS). To initiate the treatment cycle, the patient received intravaginal progesterone at night for 10 days to induce a withdrawal bleed. Oocyte retrieval was performed on day 11 following a withdrawal bleed. The patient was administered 10,000 IU of HCG subcutaneously 36 h prior to oocyte collection. A total of 63 immature oocytes were obtained; 10 were morphologically abnormal. Following incubation for 24--48 h in the maturation medium, TC-199 supplemented with 20% patient's own serum, 75 mIU/ml FSH and LH, 77.4% (41/53) of the oocytes were at the metaphase-II stage. Thirty-one (31/41, 75.6%) were fertilized using ICSI with her husband's spermatozoa, 15 fertilized oocytes were cultured for embryo transfer and 16 were frozen at the pronuclear stage. Pregnancy ensued following fresh embryo transfer. Unfortunately, the pregnancy was miscarried eight weeks later. However, the second frozen-thawed embryo transfer attempt resulted in a full-term pregnancy with delivery of a healthy male infant.     

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.     

Endocrinology: A prospective randomized single-blind comparative trial of nafarelin acetate with buserelin in long-protocol gonadotrophin-releasing hormone analogue controlled in-vitro fertilization cycles

The use of gonadotrophin-releasing hormone (GnRH) agonists tocontrol ovulation induction cycles for in-vitro fertilization(IVF) has been shown to increase the pregnancy rate and livebirth rate compared with non-analogue cycles. Different formulationsof GnRH agonist are available with different routes and frequenciesof administration. In this prospective study, the efficacy andsafety of intranasal nafarelin and buserelin as adjunctive therapyduring IVF were assessed. A total of 240 female patients wererecruited to the study and in the first phase patients wererandomized to receive either intranasal nafarelin 200 µgtwice daily or buserelin 200 µg five times daily. In thesecond phase, patients received either nafarelin 400 µgtwice daily or buserelin 200 µg five times daily. Nafarelin400 µg and buserelin 200 µg both produced clinicalpregnancy rates of 31% per recruit and 39% per embryo transfer.The rates for nafarelin 200 ug were 23 and 37% respectively.There was no statistically significant difference in pregnancyrates, by either drug or dosage. The time taken for pituitarydown-regulation to be achieved was significantly longer on nafarelin200 µg than on either nafarelin 400 µg or buserelin.The total number of days stimulation with human menopausal gonadotrophinrequired to reach criteria for human chorionic gonadotrophin(HCG) administration was significantly longer on buserelin thanon either dose of nafarelin. Median serum oestradiol concentrationson the day of HCG administration were significantly higher oneither dose of nafarelin than on buserelin.     

Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome: Case report

This case report describes an ongoing pregnancy after cryopreservation of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome (PCOS). Oocyte retrieval was performed on day 18. The patient was administered 10 000 IU of hCG s.c. 36 h prior to oocyte collection. A total of 61 immature oocytes was obtained. Following incubation for 24-72 h in the YS maturation medium supplemented with 30% follicular fluid (hFF), 1 IU/ml FSH, 10 IU/ml hCG and 10 ng/ml rhEGF, 65.6% (40/61) of the oocytes were at the metaphase II stage. Thirty-eight oocytes (38/40, 95.0%) were fertilized after ICSI with the patient's husband's sperm and the 2PN oocytes were co-cultured with cumulus cells in YS medium supplemented with 10% hFF. Four embryos were transferred into the uterus on day 4 following oocyte retrieval but this failed to result in pregnancy. Eight embryos were developed to expanded blastocyst stage. The blastocysts were vitrified on electron microscope grids. Two years after cryopreservation, four blastocysts were thawed, three re-expanded and these frozen-thawed blastocysts were transferred to the uterus. A viable twin pregnancy was confirmed by ultrasound scan.     

Rescue ICSI of unfertilized oocytes after IVF

BACKGROUND: Failed fertilization after IVF occurs in 10-20% of cycles. Conflicting results of rescue fertilization by ICSI have been reported. We therefore compared the success rate in terms of fertilization and pregnancy of cycles in which rescue ICSI was performed with those from a matched control group of primarily ICSI cycles. METHODS: Unfertilized oocytes from IVF cycles with total fertilization failure where at least four metaphase II oocytes were available were treated by ICSI (group I; n = 120). A matched control group was established with patients undergoing ICSI during the same period (group II; n = 280). RESULTS: Both fertilization rate and the proportion of embryos with four blastomeres on day 2 after ICSI were significantly higher in the control group (P < 0.05). Embryo quality, however, was comparable in both groups. The pregnancy rate in the control group was 25.3% whereas in group I with rescue ICSI, no pregnancy was obtained. CONCLUSIONS: Although unfertilized oocytes after IVF can be fertilized by ICSI, the developmental potential of the ensuing embryos is very poor. Therefore, rescue ICSI after total failure of fertilization is not recommended.     

Endocrinology: The induction of ovulation with pulsatile gonadotrophin-releasing hormone (GnRH) administration in hyperandrogenic women after down-regulation with buserelin or suppression with an oral contraceptive

The hypothalamic—pituitary axis of 22 hyperandrogenicinfertile women had suppression with either the gonadotrophin-releasinghormone (GnRH)-analogue buserelin (n = 12) or with an oestrogen—gestagencompound (Diane^®; ⁿ = 12). This was followed by pulsatileGnRH application for inducing ovulation (Zyklomat^®). Interms of ovulation and pregnancy rates the buserelin pre-treatmentwas more effective than the steroid pre-treatment, especiallyin hyperandrogenic non-polycystic ovaries (PCO).     

Cytogenetic analysis of living human oocytes: cellular basis and developmental consequences of perturbations in chromosomal organization and complement

Chromosome complement and location were examined by fluorescencemicroscopy for 225 melotically mature (metaphase II) human oocytesafter staining with DNA-specific probes. Both preovulatory oocytesand oocytes that failed to fertilize in vitro were analysed.After inspection in the living state, oocytes were selectedfor karyotyping or transmission electron microscopy. The findingsdemonstrate a high correlation between assessments of chromosomecomplement in living oocytes and the results from subsequentkaryotypes. In addition to numerical aberrations (aneuploidy),the results also demonstrate the ability to detect abnormalitiesin chromosome structure and distribution. Specifically, thisapproach identified living oocytes that (1) contained no apparentchromosomes in the ooplasm, (2) contained chromosomes not associatedwith the MI! spindle and (3) had weak or no detectable chromosomalfluorescence In the first polar body. The findings demonstratethat 8% of the oocytes were aneuploid (hypohaploid or hyperhaplold).Another 6.5% displayed anomalies in chromosome structure ordistribution that could lead to aneuploid situations. The resultsare discussed with respect to the origin, occurrence and developmentalconsequences for such oocytes.     

Morphological and cytogenetic observations of unfertilized human oocytes and abnormal embryos obtained after ovarian stimulation with pure follicle stimulating hormone following pituitary desensitization

Morphological and cytological observations of 189 unfertilizedoocytes and 40 abnormal embryos obtained from 32 patients ina routine in-vitro fertilization programme were performed. Boththe oocytes and the embryos were mounted whole to preserve theoriginal topology of all the structural elements. With the appliedprotocol of ovarian stimulation associating pituitary desensitizationand follicle stimulating hormone stimulation, a high degreeof immaturity of the unfertilized eggs was observed in comparisonwith previous reports. This immaturity was deduced from thehigher incidence of unfertilized eggs arrested at the germinalvesicle or metaphase I stage, as well as metaphase II oocyteswith multiple metaphase plates. Nine triploid and four tetraploidembryos were analysed: except for one tetraploid embryo, allthe polyploid embryos cleaved. The percentages of mononucleatedblastomeres in these polyploid embryos were 57 and 27% respectively.We also analysed 21 cleaving diploid embryos which exhibiteda high degree of fragmentation.No more than 40% of the blastomerescontained single nucleus. Moreover, in only one of the 21 diploidembryos could all the blastomeres be considered normal.     

Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group

In this prospective and randomized study, 188 patients received the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix, and 85 patients the LHRH agonist buserelin to prevent endogenous luteinizing hormone (LH) surges during ovarian stimulation in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Ultimately, 181 patients (96.3%) in the cetrorelix group, and 77 (90.6%) in the buserelin group, reached the day of the human chorionic gonadotrophin (HCG) injection. The mean number of human menopausal gonadotrophin (HMG) ampoules administered and the mean number of stimulation days with HMG were significantly less in the cetrorelix group than in the buserelin group (P < 0.01). A rise in LH and progesterone concentrations was observed in three of the 188 patients (1.6%) who received cetrorelix. On the day of the HCG administration, more follicles of a small diameter (11-14 mm) were observed in the buserelin group than in the cetrorelix group (P = 0. 02) and the mean serum oestradiol concentration was significantly higher in patients who received buserelin than in those who received cetrorelix (P < 0.01). Similar results were observed in fertilization, cleavage and pregnancy rates in the two groups. In conclusion, the use of the LHRH antagonists might be considered more advantageous because of the short-term application needed to inhibit gonadotrophin secretion, so allowing a reduction in the treatment time in a clinically significant manner.     

Chromosome analysis of human oocytes failing to cleave after insemination in vitro

Seventy-six human oocytes lacking signs of fertilization 48h after insemination in vitro were fixed for chromosome analysis.Only one out of 61 oocytes showed mitotic chromosomes indicatingfertilization. Thus, non-cleavage is almost synonymous withnon-fertilization. Chromosomes in second meiosis were foundin 60 oocytes, of which 52 (86.7%) were analysable. Of these,only 18 (34.6%) displayed an apparently normal 23,X karyotype.Fifteen preparations showed no chromosomes at all but some containedsmall nuclei, most likely due to degeneration. An earlier stageof degeneration could be represented by a group of second meiosisoocytes (20.0%) carrying between 10 and 18 chromosomes. Thelarge proportion of chromosome abnormalities in this samplemight have been due to the fact that the oocytes were unfertilizedand perhaps that abnormal oocytes had matured due to ovarianstimulation.     

Fertilization and early embryology: A retrospective analysis of unfertilized and presumed parthenogenetically activated human oocytes demonstrates a high frequency of sperm penetration

A total of 518 normal-appearing, meiotically mature human oocytesthat were judged unfertilized after insemination in vitro wereexamined for sperm penetration by conventional fluorescenceand laser scanning confocal microscopy with DNA-specific probes.A similar analysis was performed on 29 single pronuclear oocytesthat were presumed to originate by spontaneous (parthenogenetic)activation. The results demonstrate that 22% of the unfertilizedoocytes and 52% of the presumed parthenogenetic oocytes wereactually penetrated. Sperm penetration occurred in both normozoo-spermicand male factor cases. The findings indicate the importanceof penetration analysis indetermining the causes of fertilizationfailure that may reside with the male or female gamete, especiallywhen assessing the utility of and necessity for assisted fertilizationin subsequent attempts. The results also suggest that the cytoplasmiccapacity to decondense sperm DNA may decline more rapidly thanthe ability of the oocyte to be penetrated and to mount an effectiveblock to polyspermy.     

Is continuation of a gonadotrophin-releasing hormone agonist (GnRHa) necessary for women at risk of developing the ovarian hyperstimulation syndrome?

A total of 28 women scheduled for in-vitro fertilization used buserelin and human menopausal gonadotrophin (HMG) for ovarian stimulation. One group (I) of 17 women was given human chorionic gonadotrophin (HCG 10,000 IU) to trigger ovulation, but the resulting embryos were electively cryopreserved because of the risk (serum oestradiol greater than or equal to 3500 pg/ml) of developing the ovarian hyperstimulation syndrome (OHSS). Six women continued the buserelin therapy in the luteal phase and eleven did not. In group II (n = 11), the HMG injections were discontinued because of an exaggerated ovarian response and the HCG was omitted. Six of these women continued the buserelin injections until the onset of menses and five did not. In both groups, the ovarian response to induction of ovulation (serum oestradiol concentrations and number of follicles) was similar for those who did or did not continue buserelin therapy. There was no difference in the rate of ovarian quiescence (weekly fall in serum oestradiol concentration following the stimulation) between those women who did or did not continue the buserelin therapy in either group. The serum luteinizing hormone concentrations remained low in all women in both groups. We conclude that the omission of buserelin therapy after discontinuing the HMG in women at risk of developing OHSS does not affect subsequent ovarian quiescence.     

The use of a short regimen of buserelin, a gonadotrophin-releasing hormone agonist, and human menopausal gonadotrophin in assisted conception cycles

The outcome of in-vitro fertilization treatment using buserelin, an agonist of luteinizing hormone releasing hormone, given in a short stimulation regimen with human menopausal gonadotrophin (HMG), was compared with a conventional regimen including clomiphene citrate (CC). A total of 94 infertile women underwent cycles of treatment with intranasal buserelin, 500 micrograms daily from the first day of menstruation and also HMG daily from the third day. The same patients had previously undergone unsuccessful treatment cycles with CC and HMG. Overall, addition of buserelin resulted in fewer cycles being abandoned (10 versus 34%) and none of the patients ovulated prior to collection. The mean total dose of HMG required was increased by 74% in buserelin cycles. Significantly more oocytes were collected with buserelin treatment (mean 5.9 versus 4.4, P less than 0.01) and, thus, significantly more embryos were transferred (mean 2.3 versus 1.2, P less than 0.0001) although fertilization and cleavage rates were unchanged. Fifteen pregnancies were achieved, giving a clinical pregnancy rate of 22% per embryo transfer. These pregnancies resulted in 16 live births (7 singletons, 3 twins, 1 triplets). Four pregnancies failed before 14 weeks gestation. We conclude, therefore, that the substitution of buserelin for CC for ovarian stimulation in poor responders results in an improved outcome, both in terms of the number of oocytes collected and the pregnancy rate per treatment cycle.     

Endocrine responses to gonadotrophins after LHRH agonist administration on cycle days 1-4: prevention of premature luteinization

A treatment regime comprising an intranasally administered luteinizinghormone-releasing hormone (LHRH) agonist analogue (buserelin)on cycle days 1–4, followed by gonadotrophin administration[follicle stimulating hormone (FSH)/human menopausal gonadotrophin(HMG)] resulted in identical oestradiol (E₂) responses comparedwith the reference method using clomiphene citrate (CC) andgonadotrophins. Immediately after analogue administration (day4), buserelin-treated women showed short-lived elevations inserum LH and progesterone concentrations, but in the later follicularphase, the serum LH concentration was lowered compared withthe controls. None of the women treated with analogue displayedelevated serum LH or progesterone concentrations at the timeof injection of human chorionic gonadotrophin. In the earlyluteal phase, these women had higher serum levels of progesteroneand higher progesterone to E2 ratios than the controls, butthe length of the luteal phase was slightly shortened. Hence,in hyperstimulated cycles, 4-day treatment with buserelin causedprofound endocrinological changes: namely, short-term rescueof the corpus luteum, prevention of an endogenous LH rise andpremature luteinization and increased progesterone productionin the early luteal phase