Efficacy of oocytes donated by older women in an oocyte donation programme

Population and insemination studies indicate that women experiencedeclining fertility with ageing. The question therefore ariseswhether older women are suitable oocyte donors. This study addressesthis issue by examining the relationship between oocyte donorage and clinical outcome in a large oocyte donation programme.We retrospectively reviewed data from 458 consecutive oocytedonation cycles completed by 164 different designated oocytedonors. Data were divided into two groups: group A, cycles withdonors aged 21–30 years at the time of follicular aspiration(193 cycles, 88 donors); and group B, cycles with donors aged31–40 years at the time of follicular aspiration (265cycles, 86 donors). Five donors, because of ageing during repetitivedonations, contributed data to groups A and B. In a given cycle,all oocytes for a recipient came from only one designated donor.Comparing the two donor groups, there was no difference in theamount of gonadotrophin used to achieve optimal stimulation;however, more oocytes were obtained from group A than groupB donors (16.8 ± 6.9 and 15.1 ± 8.1 respectively,P < 0.05). Similar percentages of oocytes were fertilizedin each group, resulting in the transfer of comparable numbersof embryos (4.5 ± 1.1 and 4.4 ± 13 respectively).Comparable clinical pregnancy rates were achieved (group A,36%; group B, 37%). The spontaneous abortion rates were alsosimilar (group A, 20%; group B, 12%), resulting in comparableongoing and delivered pregnancy rates per cycle (group A, 29%;group B, 32%) and per embryo transferred (group A, 6.4%; groupB, 7.3%). In conclusion, women of proven fertility should notbe excluded from donating oocytes simply because of their age.There exists a cohort of fertile women who resist the decreasingfecundity and increasing spontaneous abortion rates associatedwith ageing. With careful screening, many women of proven fertilitycan donate oocytes until the age of 40 years with an efficacyequal to that of younger women. Given the relative shortageof suitable oocyte donors, and increasing requests from recipientswith previous donor oocyte babies to obtain oocytes from thesame, now older, donor, the findings of this study are of practicalclinical importance.     

Infertility: Oocyte donation: comparison between recipients from different age groups

Oocyte donation was carried out in 87 patients in 141 replacementcycles. These patients received oocytes from 108 women undergoingassisted reproductive technology procedures at our centre. Standardizedhormonal replacement therapy and in-vitro fertilization procedureswere performed. We divided recipients into four groups accordingto their age (group A, 21–35 years; B, 36–40 years;C, 41–49 years; and D, 50–61 years). Oocytes donorswere 21–35 years old, and equally spread across thesedifferent age groups. There were significant differences inthe pregnancy and implantation rates according to the age ofthe recipients; which were 45% and 23% respectively in women21–35 years old (group A) versus 23% and 10% in women41–49 years old (group C). A comparison of data betweenoocyte donors and their specific recipients showed similar resultsin donors and young recipients, with pregnancy rates of 45%and 42% and implantation rates of 23% and 19.5% respectively.Statistically significant differences were found between donorsand the older recipients, pregnancy rates being 43% versus 23%,and implantation rates 18% versus 10%. These data seem to demonstratea lesser likelihood of pregnancy and implantation in older recipientsbecause of increasing uterine age.     

The effects of cooling human oocytes

Preovulatory human oocytes were cooled to 0°C at 1°C/minwith or without the cryoprotectant dimetbyl suiphoxide (DMSO),to assess the effects of cooling on the meiotic spindles andon oocyte structure. Batches of oocytes, cultured for 3–9h, were held at 0°C for 20 or 60 min and then fixed fortransmission electron microscopy (TEM) either at 0 or 8°C.Control oocytes were not cooled and were fixed at 22 or 37°Cfor comparison. TEM revealed that 80% of the oocytes were atmetaphase II, while 20% were at metaphase I and most had resumedmeiosis recently. Control oocytes had more or less barrel-shapedmeiotic spindles composed of microtubules (MT), some associatedwith chromosomes at kinetochores. Both metaphase I and II spindleswere disassembled when cooled and fixed at 0°C, with orwithout DMSO, due to extensive depolymerization of MT. The fewMT that survived were found at the poles or were bundled togetheror were associated with chromosomes. Kinetochores were not prominent.Some oocytes cooled with DMSO and fixed at 0 or 8°C showedevidence of MT, but the spindles were still disorganized andwere abnormal In structure. Chromosomes tended to dump togetheror were dislocated in the cortical ooplasm in cooled oocytes,but widespread scattering was not observed. This was particularlyevident in the absence of DMSO. Elements of the endoplasmicreticulum, Golgi, mitochondria and the cytosol were also adverselyaffected In some of the cooled oocytes and their surroundingcumulus cells. The results show that meiotic spindles are verysensitive to simple cooling and that DMSO does not provide substantialstabilization of the meiotic spindle even at 0/C. The findingsare discussed with reference to recent work on frozen humanand mouse oocytes.     

Female age predicts embryonic implantation after ICSI: a case-controlled study

From 1 October 1991 until 31 December 1993, 1270 cycles forintracytoplasmic sperm injection were performed. Of these, 71(5.6%) were carried out in women 40 years of age. The semencharacteristics in couples40 years of age or <40 years weresimilar. The mean male age for the older group of women was47.1 years (range 34–67) versus 35 years (range 25–71)for the younger group of women (P <0.001). The mean femaleage was 41.9 years (range 40–-47) and 31.8 years (range23–39). The numbers of cumulus—oocyte complexesand metaphase-II oocytes were significantly lower in women 40years of age (P < 0.001). The mean numbers of replaced embryoswere respectively 2.3 (133/59) in women 40 years of age and2.5 (160/63) in women <40 years of age. The delivery rateper retrieval and per transfer was significantly lower in women40 years of age (P < 0.05). The delivery rates per retrievaland per transfer were respectively 7% (5/71) and 8.5% (5/59)in the older group of women versus22.5% (16/71) and 25.4% (16/63)in the younger group. Female age is the predictive factor forembryonic implanatation.     

Features associated with reproductive ageing in female rhesus monkeys

BACKGROUND: The specific aims were to determine the effects of maternal age on the meiotic and developmental competence of oocytes and incidence of chromosomal anomalies in oocytes from a population of fertile rhesus monkeys. METHODS: Monkeys were divided into two age groups (4-15 and 16-26 years of age) and underwent ovarian stimulation for collection of oocytes. RESULTS: In the older, compared with younger, monkeys, serum basal concentrations of FSH were elevated (P < 0.05), peak concentrations of estradiol during a stimulation cycle were diminished (P < 0.05), and mean numbers of oocytes retrieved following ovarian stimulation were markedly (P < 0.05) reduced. There were no significant maternal age-related impairments in oocyte maturation, fertilization or blastocyst development. Both abnormal numbers of whole chromosomes, as well as free chromatids, were detected in a limited number of rhesus oocytes. CONCLUSIONS: Similarities between female rhesus monkeys and women in several features associated with reproductive ageing, in conjunction with our ability to perform IVF and other assisted reproductive techniques in monkeys, demonstrate the suitability of these animals for studies on human reproductive ageing and maternal age-related infertility. Although maternal age-related impairments in oocytes were not evident prior to implantation, further studies may reveal more subtle impairments, manifested during post-implantation development.     

Spermatogenesis and meiotic chromosomal behaviour in aged men

Spermatogenesis and meiotic chromosomal behaviour was analysedin a group of males aged 60–80 years. Decreased spermatogenesiswas found in most of these aged men although individual differencesexisted. Chromosomal analysis of cells undergoing diakinesisshowed significantly lower chiasma frequency in elderly men,compared with a group of younger control males and also withinfertile males.     

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

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

Chromosome anomalies in human oocytes failing to fertilize after insemination in vitro

Three-hundred-and-two unfertilized oocytes left over from successfulin-vitro fertilization (IVF) attempts in 143 women (27–42years) on a follicular stimulating hormone-human menopausalgonadotrophin (FSH-HMG) stimulation regime were subjected tochromosome analysis. Ten oocytes were degenerated with no visiblechromosomes and 41 metaphases had chromosomes that were clumpedtogether which could not be interpreted either numerically orstructurally. Of the remaining oocytes, 76.6% (192/251) hada normal haploid complement (n = 23), 13% (33/251) were hypohaploid(n = 19–22), 8% (20/251) were hyperhaploid (n = 24–26),2% (5/251) were diploid (2n = 46) and 0.4% (1/251) had structuralrearrangements. The 21% aneuploidy was from 24 different patientsand hypohaploid sets had chromosomes missing mainly from theA, B, C, D and G groups while the hyperhaploid sets had extrachromosomes from A, B, D, G and E groups of the human karyotype.The mean age of patients showing aneuploid oocytes was 36.7years which was above the mean for the entire group. The aneuploidymay have been brought about by errors in oogenesis (anaphaselagging or non-disjunction) and may offer one explanation forfertilization failure and overall low pregnancy rates afterIVF.     

Fertilization and early embryology: Increase in the rate of diploidy with maternal age in unfertilized in-vitro fertilization oocytes

Human oocytes which failed to fertilize in vitro were fixedfor cytogenetic analysis. Metaphase II chromosomes were identifiedin 286 oocytes, of which 233 were suitable for cytogenetic analysis.In all, 181 oocytes had a normal haploid karyotype (77.7%),while the remaining 52 were abnormal (28 aneuploid, 14 diploidand 10 tetraploid). The rate of aneuploidy did not increasewith maternal age. However, the proportion of diploid oocytesincreased significantly with advancing maternal age (P <0.01), being particularly obvious in women aged >35 years.     

Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte

At ovulation, the mouse oocyte is arrested at metaphase of thesecond meiotic division. Since microtubules are thermo-and chemosensitivestructures, the effects of 1.5 M dlmethylsulphoxide and 1.5M 1,2-propanediol were studied at room temperature on the morphologyof the meiotic spindle. Oocytes incubated at 37°C or atroom temperature served to estimate the effect of temperaturein the experiment. The meiotic spindle was visualized by immunogold-silverstaining of microtubules. In the control group at 37°C,88% of oocytes had normal spindles. After incubation at roomtemperature for the same time, 89% of oocytes showed abnormalspindles. In the oocytes exposed to dimethyl sulphoxide or 1,2-propanediolat room temperature a protective effect on spindle morphologycould be recognized. Subsequent incubation at 37°C resultedin partial restoration of the observed abnormalities after coolingto room temperature and after exposure to dhnethytsulphoxide.incubation at 37°C after exposure to 1,2-propanediol atroom temperature induced spindle absence in the majority ofoocytes. Although this latter condition allowed fertilizationwithout increased incidence of ploidy abnormalities, a rolefor 1 as an activating agent is hypothesized.     

Ovary and ovulation: DNA fragmentation of oocytes in aged mice

To investigate whether female fertility decreases with age dueto poor oocyte quality, we examined the presence of DNA fragmentationin ovulated oocytes from young, mature and aged mice. Oocytesfrom three age groups of female mice (7–8, 20–24and 40–48 weeks) were retrieved from the ovlducts 15 hafter human chorionic gonadotrophin (HCG) injection. Oocytesfrom each mouse were incubated in a CO₂ Incubator for 0–60h in human tubal fluid (HTF). After incubation, each oocytewas stained with the terminal deoxynucleotidyl transferase-mediateddUDP nick-end labelling (TUNEL) method. The rate of DNA fragmentation(interpreted as apoptotic changes) was significantly higherfor oocytes from aged mice, and the fertilization rate was significantlylower, compared with oocytes from young and mature mice. Ourresults suggest that DNA fragmentation of oocytes might be oneof the reasons for poor oocyte quality and lower fertility inthe aged group.     

Chromosome anomalies in human oocytes in relation to age

Cytogenetic analysis of oocytes remaining unfertilized afterin-vitro fertilization showed that the source of data obtainedcould be divided into degenerating and ‘healthy’oocytes. The degenerating oocytes, which showed different degreesof chromosome breakage, accounted for a quarter of the total.They were found in older patients with a mean age of 35.0 years.The healthy oocytes without chromosome breaks were mostly haploidand fell into two main groups, those with a normal MII, 23,X chromosome complement, and those abnormal in which singlechromatids replaced a whole chromosome. No oocytes hyperhaploidfor an extra whole chromosome were found. We hypothesize thatthe single chromatids at second meiotic metaphase arise by precociousdivision of chromosome univalents at anaphase I (predivision)and that this may be the major mechanism for trisomy formationin man, rather than the non-disjunction of whole bivalents asgenerally assumed.     

Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen--thawed mouse oocytes

Mouse oocyte—cumulus masses were added to 1.5 dimethylsulphoxide (DMSO) + 20% fetal bovine serum (FBS) that had beenprecooled at +4°C, were frozen by slow cooling to an intermediatetemperature of –60°C before being plunged into liquidnitrogen at –196°C, subjected to a controlled thaw,expelled into 1.5 M DMSO + FBS at 4°C, and then washed inmedium + FBS at 37°C. Of 7733 oocytes treated, 78.4% wereviable (controls; no treatment: 94.2% of 2764 oocytes; cryoprotectantonly: 92.2% of 2991 oocytes). The oocyte losses were not dueto complete loss of all oocytes from some straws or mice, sinceanalysis of individual straws containing oocytes from a singlemouse revealed considerable inter-straw/mouse variation. Amongstsurviving oocytes, no significant differences between frozenand control oocytes in spindle, chromosomal or microfilamentorganization were recorded. Two significant differences wereobserved: (i) fewer frozen—thawed oocytes had zonae resistantto chymotrypsin digestion, and (ii) spindle organization incontrol oocytes, but not frozen—thawed oocytes, was improvedby 3 h incubation at 37°C. More of the abnormal than thenormal frozen—thawed and control oocytes were surroundedby zonae which were resistant to digestion by chymotrypsin.     

Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.     

Fertilization and early embryology: The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection

Implementation of intracytoplasmic sperm injection (ICSI) inhuman in-vitro fertilization (IVF) has highlighted the needfor information about the risk of nuclear spindle damage causedby this procedure. For this purpose we studied the final productsof oocyte meiosis at the first cleavage division of multipronuclearzygotes arising after ICSI, and compared the results with abnormallyfertilized oocytes after conventional in-vitro insemination.Of 37 successfully analysed tripronuclear zygotes, 18 had threeindividual metaphases. Abnormal complements of 11 zygotes inthis group indicated that non-disjunction occurred predominantlyat the second meiotic division of the oocytes. Nine of the 37tripronuclear zygotes exhibited two individual metaphases. Sevenwere abnormal and there were some indications that non-disjunctiontook place during oocyte meiosis. Of the 37 tripronuclear zygotes,10 had a single metaphase and three showed an aneuploid numberof chromosomes. The overall rate of aneuploidy among tripronuclearmicroinjected zygotes was 56.7%. In addition, seven zygoteswith more than three pronuclei arising after ICSI displayedseverely depleted chromosome complements. The incidence of non-disjunctionin oocytes fertilized by conventional in-vitro inseminationwas significantly lower (20.0%, P < 0.01), since only fourzygotes had an aneuploid number of chromosomes. Our findingssuggest that ICSI might interfere with regular chromosome segregationat the second meiotic division of the oocytes.     

The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary

The human oocyte appears to be particularly prone to meiotic errors, and the incidence of these errors is strongly influenced by maternal age. We have initiated studies of human oocytes from unstimulated ovaries and have observed age-related effects on the meiotic process in oocytes from unselected antral follicles. Specifically, in oocytes obtained from donors over the age of 35 years, the majority of oocytes that extruded a first polar body in culture and arrested at second meiotic metaphase had aberrations in spindle formation and chromosome alignment. Similarly, observations of a limited number of oocytes at first meiotic metaphase suggest disturbances at this stage of meiosis as well. Finally, preliminary results of non-disjunction studies suggest that the frequency of errors in chromosome segregation at the first meiotic division is influenced by donor age in in-vitro matured oocytes as it is in oocytes undergoing meiotic maturation in vivo. These data provide direct evidence that the meiotic competence of oocytes from unstimulated ovaries declines with donor age. Similarly, studies of in-vitro fertilization (IVF) pregnancies in older women indicate that the developmental competence of the human oocyte declines with age. Since both meiotic and developmental competence are acquired during the late stages of oocyte growth, we postulate that an age- related decline in the process of folliculogenesis results in reduced oocyte quality and that the well characterized age-related increase in meiotic non-disjunction is one symptom of compromised oocyte growth.      

Spindle imaging: a new marker for optimal timing of ICSI?

BACKGROUND: The LC Polscope facilitates visualization of the meiotic spindle in human oocyte. This study aimed to investigate meiotic spindle assembly in correlation to time elapsed after HCG administration, and to determine whether spindle imaging may serve to indicate the likelihood of fertilization and embryo cleavage. METHODS: Metaphase II (MII) oocytes from 103 couples who were being treated for male infertility were imaged with the Polscope prior to sperm injection. Spindle imaging was correlated to time elapsed from HCG administration, fertilization rate and embryo cleavage. The main outcome measures were spindle visualization, fertilization and embryo cleavage on day 3. RESULTS: A total of 770 MII oocytes were imaged. A spindle was imaged in a significantly higher number of oocytes from >or=38 h after HCG administration compared with those in the <38 h group (78.1-81.5% versus 61.6%; P < 0.001). The fertilization rate in oocytes with a visible spindle was statistically higher compared with oocytes in which spindle could not be detected (70.4% versus 62.2%; P = 0.035). We found no relationship between spindle imaging and embryo cleavage on day 3. CONCLUSIONS: Spindle imaging, in addition to first polar body appearance, is an accurate indicator for oocyte maturity. We suggest that spindle imaging be performed prior to sperm injection.     

影响高龄小鼠卵母细胞质量的超微结构因素

目的 探讨高龄小鼠卵母细胞线粒体、皮质颗粒、微丝、纺锤体及染色体形态和排布的变化规律。 方法 将雌性昆明小鼠分为年轻组（4～8周）和高龄组（48～52周）。腹腔内注射孕马血清促性腺激素（PMSG）10 IU/只行促排卵,48 h后腹腔内注射人绒毛膜促性腺激素（hCG）10 IU/只,14 h后收集输卵管的卵冠丘复合物(OCCC)。脱去颗粒细胞,获取成熟卵母细胞（MⅡ期）经2%多聚甲醛固定。抗小鼠α-tubulin单克隆抗体染色卵母细胞纺锤体的微管,罗丹明标记的鬼笔环肽染色微丝,FITC结合的小扁豆凝集素染色皮质颗粒,Mito Tracker Green FM染色线粒体。激光扫描共焦显微镜下观察。 结果 共获取小鼠卵母细胞372枚,其中年轻组179枚,高龄组193枚。高龄组小鼠卵母细胞纺锤体异常率（71.78% ±1.27%）、染色体排列异常率（65.16% ±1.52%）明显高于年轻组（18.93% ±1.27%,32.24% ±1.10%）（P<0.01,P<0.01）;高龄组小鼠卵母细胞线粒体的荧光强度（28.09±6.62）明显低于年轻组（45.07 ±5.90）（P<0.01）;高龄组小鼠成熟皮质颗粒（Ⅲ级皮质颗粒）（51.00%±100%）明显低于年轻组（94.04% ±0.71%）（P<0.01）。 结论 高龄小鼠卵母细胞中各主要细胞器、细胞骨架及染色体排列异常率明显增加,可能是导致高龄受孕率低的重要原因。     

女性年龄对卵母细胞纺锤体和染色体构型的影响

目的探讨女性年龄对卵母细胞纺锤体和染色体构型的影响。方法取试管婴儿术后卵龄1天的未受精卵母细胞,采用免疫细胞化学和激光共聚焦术检测卵母细胞纺锤体和染色体构型。结果25~29岁女性卵母细胞Ⅰ级纺锤体和染色体比率分别为33%和31%,显著高于30~34岁(P<0.05)和35~40岁女性(P<0.01);而25~29岁女性卵母细胞的Ⅲ级纺锤体和染色体率均为54%,明显低于30~34岁(P<0.01)和35~40岁女性(P<0.05)。结论卵母细胞纺锤体和染色体构型异常率与女性年龄存在明显的相关性。