Aneuploidy In Oocytes Research Articles

Male mouse meiotic chromosome cores deficient in structural proteins SYCP3 and SYCP2 align by homology but fail to synapse and have possible impaired specificity of chromatin loop attachment

The targeted deletion of the meiotic chromosome core component MmSYCP3 results in chromosome synaptic failure at male meiotic prophase, extended meiotic chromosomes, male sterility, oocyte aneuploidy and absence of the MmSYCP2 chromosome core component. To test the functions of SYCP2 and SYCP3 proteins in the cores, we determined the effect of their deletion on homology recognition by whole chromosome painting and the effect on chromatin loop attachment to the cores with endogenous and exogenous sequences. Because we observed that the alignment of cores is between homologs, it suggested that alignment is not a function of the chromosome core components but might be mediated by chromatin-chromatin interactions. The alignment function therefore appears to be separate from intimate synapsis function of homologous cores that is observed to be defective in the SYCP3^–/– males. To examine the functions of the SYCP2 and 3 core proteins in chromatin loop attachment, we measured the loop sizes of the centromeric major satellite chromatin and the organization of an exogenous transgene in SYCP3^+/+ and SYCP3^–/– males. We observed that these satellite chromatin loops have a normal appearance in SYCP3^–/– males, but the loop regulation of a 2-Mb exogenous λ phage insert appears to be altered. Normally the insert fails to attach to the core except by flanking endogenous sequences, but in the absence of SYCP2 and SYCP3, there appears to be multiple attachments to the core. This suggests that the selective preference for the attachment of mouse sequences to the chromosome core in the wild-type male is impaired in the SYCP3^–/– male. Apparently the SYCP2 and SYCP3 proteins function in the specificity of chromatin attachment to the chromosome core.

Transient exposure to the Eg5 kinesin inhibitor monastrol leads to syntelic orientation of chromosomes and aneuploidy in mouse oocytes

Aneuploidy may result from abnormalities in the biochemical pathways and cellular organelles associated with chromosome segregation. Monastrol is a reversible, cell-permeable, non-tubulin interacting inhibitor of the mitotic kinesin Eg5 motor protein which is required for assembling and maintaining the mitotic spindle. Monastrol can also impair centrosome separation and induce monoastral spindles in mammalian somatic cells. The ability of monastrol to alter kinesin Eg5 and centrosome activities and spindle geometry may lead to abnormal chromosome segregation. Mouse oocytes were exposed to 0 (control), 15, 30, and 45μg/ml monastrol in vitro for 6h during meiosis I and subsequently cultured for 17h in monastrol-free media prior to cytogenetic analysis of metaphase II oocytes. A subset of oocytes was cultured for 5h prior to processing cells for meiotic I spindle analysis. Monastrol retarded oocyte maturation by significantly (P<0.05) decreasing germinal vesicle breakdown and increasing the frequencies of arrested metaphase I oocytes. Also, significant (P<0.05) increases in the frequencies of monoastral spindles and chromosome displacement from the metaphase plate were found in oocytes during meiosis I. In metaphase II oocytes, monastrol significantly (P<0.05) increased the frequencies of premature centromere separation and aneuploidy. These findings suggest that abnormal meiotic spindle geometry predisposes oocytes to aneuploidy.

Thirteen years' experience of preimplantation diagnosis: report of the Fifth International Symposium on Preimplantation Genetics

Preimplantation genetic diagnosis (PGD) has been further developed into a practical option for avoiding the birth of affected children, representing an important complement to traditional prenatal diagnosis. More than 1000 unaffected children have been born after PGD, suggesting the accuracy and safety of the procedure, which is currently also used with the establishment of potential donor progeny for stem cell treatment of siblings. Together with progress in the establishment of embryonic stem (ES) cells, this may contribute to the development and application of stem cell therapy. The accumulated experience of thousands of PGD cycles for poor prognosis IVF patients provides further evidence of the improvement of clinical outcome, particularly obvious from the reproductive history of PGD patients. A high prevalence of aneuploidies in oocytes and embryos may affect the accuracy of PGD for single gene disorders, making aneuploidy testing an important part of PGD for causative genes and preimplantation human leukocyte antigen (HLA) typing. A sequential sampling of both oocytes and the resulting embryos may improve accuracy of aneuploidy testing and may also allow the detection and avoidance of transfer of embryos with uniparental disomies. Current developments and application of nuclear transfer and sperm duplication techniques, and microarray technology, may also contribute to the improvement of PGD and help in the development of PGD for genetic expression disorders.

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (≥35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.

Perimenopausal conception.

Fertility, defined as the ability to achieve a pregnancy, declines gradually over the woman's lifespan. Although this decline seems to begin from the age of 30 years, it is more obvious between 35 and 40 and increases dramatically thereafter. The age of 41 is considered to be the point when fertility stops and sterility starts. The actual menopause occurs approximately 10 years after the substantial loss of conception potential. Thus, the biological rather than the chronological age of the woman can predict more accurately her fertility potential. This decline in female reproductive potential correlates with ovarian factors, although a slight contribution from the uterus itself and from the neuroendocrine axis cannot be excluded. The ovarian reserve decreases with advancing age, while a parallel decrease in the quality of the oocytes is present, as indicated by the increased incidence of oocyte aneuploidy. The endocrine function of the ovary also declines with age, the later becoming unable to sustain its normal function in the neuroendocrine axis. Additionally, the role of the various endometrial factors remains controversial. On the other hand, exposure to toxic factors and the increased prevalence of infertility-related diseases like endometriosis and PID, may also contribute. Spontaneous conception rates are minimal in perimenopausal women, mainly due to a qualitative and quantitative loss of female gametes. In the rare case of spontaneous conception achievement, complications are more likely. The application of classic ovulation induction and IVF may serve some selected cases, where the woman's ovarian biological age does not correspond to her chronological one. However, the implantation, clinical pregnancy, and live birth rates in women of advanced age undergoing IVF treatment, show very poor results. On the other hand, preimplantation genetic diagnosis is an accurate diagnostic tool for exclusion of genetically deficient embryos prior to embryo transfer. Oocyte donation seems to be the most reliable option of the perimenopausal woman, since the cumulative birth rates after four treatment cycles is approximately 80%. Cryopreservation of ovarian tissue may be an alternative in nulliparas women <40 years of age who want to have children in the future or women with the same desire who, unfortunately, have had pelvic radiotherapy, chemotherapy, oophorectomy, or premature menopause. This technique has given encouraging results in animals, but has not achieved pregnancies in humans. In the future, the use of drugs to block oocyte depletion as well as recent techniques, such as cytoplasmic or germinal vesicle transfer, will be more widely tested and may offer an option to the perimenopausal woman who wishes to conceive.

Metaphase II nuclei generated by germinal vesicle transfer in mouse oocytes support embryonic development to term.

Cytoplasmic defects are thought to cause aneuploidies in oocytes and embryos and oocyte 'reconstruction' by germinal vesicle (GV) transfer may circumvent such defects. In mice 'reconstructed' oocytes undergo meiosis and fertilize normally, but early embryonic development is compromised if their ooplasm matured in vitro. This study employs sequential MII spindle and/or pronucleus (PN) transfer to assess the embryonic potential of MII nuclei that form following GV transfer. Mouse embryos generated by these procedures were transferred to the oviducts of pseudopregnant mice to monitor pregnancy outcome. Following GV transfer, the resultant metaphase II (MII) nuclei were activated either in situ or transferred and activated in ooplasts from in-vivo matured oocytes. When exchanged with the female PN of a fertilized zygote, only the PNs that developed in in-vivo matured ooplasts generated live offspring. Viable offspring also resulted when MII nuclei were transferred to in-vivo matured ooplasts and fertilized by insemination with sperm or by artificial activation and male PN transfer. Significantly, the offspring displayed normal fertility as adults. This report of live births following GV transfer in mice illustrates the importance of the maturational history of the ooplasm at PN formation for normal embryonic and fetal development.

Mitochondrial function and cytoplasmic ability to promote germinal vesicle oocyte maturation in the mouse

Objective: Germinal vesicle transplantation (GVT) has been proposed as a possible treatment for age-related oocyte aneuploidy caused by dysfunctional ooplasm. Although the specific contribution of healthy ooplasm to normal meiosis has yet to be elucidated, impaired mitochondrial metabolism may be attributable to incomplete segregation of the oocyte chromosomes. In fact, a lower mitochondrial membrane potential is associated with an age-related higher incidence of meiotic spindle abnormality in mature human oocytes. Chloromethyl-X-rosamine (CMXRos) absorbs light energy and photosensitizes mitochondria finally leading to apoptosis. We have investigated whether mitochondrial photosensitization compromises oocyte maturation and whether GVT can rescue such mitochondria-damaged, dysfunctional oocytes.

Multi-locus (ML)-FISH is a reliable tool for nondisjunction studies in human oocytes

In the present study, we developed a fluorescence in situ hybridization (FISH) strategy, which allows a reliable determination of the chromatid number of specific chromosomes in mature human oocytes. 168 unfertilized oocytes were analyzed by dual-color FISH with two direct-labeled locus-specific DNA probes for chromosome 13 and 21. To exclude FISH failures, metaphases with abnormal signal patterns were reanalyzed by multi-locus-FISH (ML-FISH) for chromosome 13 and 21. Following dual-color FISH, abnormal signal patterns were detected in 21 out of 108 metaphases (19.4%). 17 of these metaphases were reanalyzed by ML-FISH. In contrast to the first FISH, seven metaphases showed normal signal patterns after rehybridization, whereas ten metaphases remained abnormal. Out of these real aneuploid metaphases, five showed gain or loss of a single signal (= chromatid), two showed missing double signals (= chromosome) and three showed both. In conclusion, locus-specific FISH probes facilitate differentiation between first meiotic nondisjunction of whole chromosomes and prematurely divided chromatids. Moreover, simultaneous hybridization with a second locus-specific probe on the same chromatid (ML-FISH) helps to differentiate between FISH failures and real meiotic division errors and therefore, allows a more reliable analysis of aneuploidies in human oocytes.

Aneuploidy detection in porcine embryos using fluorescence in situ hybridization

In contrast to human embryos, there are very few studies published on the frequency of chromosomal aneuploidy in farm animals. The objectives of this study were to apply a three-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in porcine embryos using chromosome-specific DNA probes, establish baseline frequencies of aneuploidy in embryos and compare the results with our previous findings of aneuploidy in spermatozoa and oocytes. The embryos were collected from superovulated gilts, which were slaughtered 48 h after insemination. FISH was performed using probes specific for the centromeric regions of porcine chromosomes 1, 10 and Y. Altogether 403 blastomeres from 114 porcine embryos were successfully investigated. Diploidy was observed in 101 (88.6%) embryos, triploidy in 2 (1.8%) embryos, mosaicism/mixoploidy in 9 (7.9%) embryos, and trisomy for chromosomes 1 or 10 in 2 (1.8%) embryos. No blastomere showed aneuploidy for chromosome Y. These findings correspond with the frequencies of aneuploidy we have found previously in porcine germ cells.

Chromosomal FISH analysis of unfertilized human oocytes and polar bodies.

The incidence of chromosomal aneuploidy was studied in 208 unfertilized metaphase II human oocytes from an in vitro fertilization program by fluorescence in situ hybridization using probes for chromosomes 18, 21, and X. Chromosome spreads were prepared by a gradual fixation-air-drying method. We obtained analyzable results from 183 oocytes and 93 polar bodies; 167 oocytes (91%) were normal, 11 (6%) were diploid, and 5 (3%) were aneuploid. Of the five aneuploid oocytes, four involved chromosome 21, and one involved the X chromosome. In this study, oocyte aneuploidy caused by both nondisjunction of bivalent chromosomes and predivision of univalent chromosomes was observed. The aneuploidy rate (9.8%) in the oocytes from women aged >==35 years was significantly higher than that (0.7%) in those aged 23 to 34 years ( P = 0.0017).

The proteasome inhibitor MG-132 induces maturation delay and aneuploidy in mouse oocytes

Objective: To test the hypothesis that temporal perturbations during oocyte maturation (OM) predispose mammalian oocytes to abnormal chromosome segregation.Design: A controlled, dose-response, in vitro study utilizing the potent, reversible, cell-permeable proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-leucinal)and mouse oocytes.Materials/Methods: Outbred, ICR mice were given PMSG 46h prior to collecting and culturing follicular oocytes in media containing 0, 5.0, 7.5, and 10.0 mcg/ml MG-132 for 6h. Oocytes were then rinsed and further cultured for 17h in MG-132-free media. Oocytes were processed and analyzed for numerical and structual chromosome aberrations.Results: Transient inhibition of proteasome-mediated proteolysis during mouse OM in vitro resulted in dose-response delays during OM and higher levels of aneuploidy. Oocytes exposed to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p <0.01) higher frequencies of MI oocytes and lower frequencies of premature centromere separation. Furthermore, the frequencies of metaphase II oocytes displaying single, unpaired chromatids and aneuploidy significantly (p <0.01) increased with MG-132 dose.Conclusions: These findings suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these data support the hypothesis tested, additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms of aneuploidy during OM.Supported by: Department of Obstetrics & Gynecology, Louisiana State University Health Sciences Center, Shreveport, LA, USA. Objective: To test the hypothesis that temporal perturbations during oocyte maturation (OM) predispose mammalian oocytes to abnormal chromosome segregation. Design: A controlled, dose-response, in vitro study utilizing the potent, reversible, cell-permeable proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-leucinal)and mouse oocytes. Materials/Methods: Outbred, ICR mice were given PMSG 46h prior to collecting and culturing follicular oocytes in media containing 0, 5.0, 7.5, and 10.0 mcg/ml MG-132 for 6h. Oocytes were then rinsed and further cultured for 17h in MG-132-free media. Oocytes were processed and analyzed for numerical and structual chromosome aberrations. Results: Transient inhibition of proteasome-mediated proteolysis during mouse OM in vitro resulted in dose-response delays during OM and higher levels of aneuploidy. Oocytes exposed to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p <0.01) higher frequencies of MI oocytes and lower frequencies of premature centromere separation. Furthermore, the frequencies of metaphase II oocytes displaying single, unpaired chromatids and aneuploidy significantly (p <0.01) increased with MG-132 dose. Conclusions: These findings suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these data support the hypothesis tested, additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms of aneuploidy during OM. Supported by: Department of Obstetrics & Gynecology, Louisiana State University Health Sciences Center, Shreveport, LA, USA.

Technical approaches to correction of oocyte aneuploidy.

This study describes the technical approaches used in treatment of age-related oocyte aneuploidy, the efficiency of each step of nuclear transplantation into mouse and human oocytes, and the ability of germinal vesicle (GV) transplantation to restore artificially induced ooplasmic damage. Finally, it examines the possibility of constructing viable female gametes by transferring diploid somatic cell nuclei into enucleated oocytes. GV stage mouse oocytes were collected from unstimulated ovaries, and human GV oocytes were donated from consenting patients undergoing ICSI. Stromal (somatic) cells were isolated from uterine biopsies of consenting patients. Mouse cumulus cells were obtained after ovarian stimulation. GV ooplasts prepared by removing nuclei were transplanted either with GV nuclei or with somatic cells by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were inseminated or analysed cytogenetically. Ooplasmic dysfunction was induced by photosensitization with a mitochondria-specific fluorescent dye. GV transplantation had an overall efficiency of 87 and 73% in the mouse and humans respectively. Maturation rates of 95 (mouse) and 64% (human) following reconstitution were comparable with those in control oocytes, as was the incidence of aneuploidy for five chromosome-specific probes after aneuploidy among the reconstituted oocytes. Photosensitization of oocytes significantly reduced the maturation rate to 4.2%, whereas 61.9% of oocytes matured after transfer of photosensitized GV karyoplasts into healthy ooplasts, with 52% of these mature oocytes being successfully fertilized by ICSI. Enucleated immature oocytes receiving mouse cumulus or human endometrial cell nuclei extruded a polar body in >40% of cases. Five out of seven successfully transferred aged human nuclei exhibited the expected number of signals with five chromosome-specific probes suggesting an appropriate chromosome separation in young ooplasm. Nuclear transplantation itself does not appear to interfere with chromosome segregation and can possibly rescue oocytes with damaged mitochondria. Finally, immature mouse ooplasm supported separation of somatic chromosomes to expected numbers, implying that haploidization may be occurring. The roles of genetic imprinting and fidelity of chromosome segregation are unknown.

Aging and infertility in women: a committee opinion

Aging and infertility in women: a committee opinion

Female Germ Cell Aneuploidy and Embryo Death in Mice Lacking the Meiosis-Specific Protein SCP3

Aneuploidy (trisomy or monosomy) is the leading genetic cause of pregnancy loss in humans and results from errors in meiotic chromosome segregation. Here, we show that the absence of synaptonemal complex protein 3 (SCP3) promotes aneuploidy in murine oocytes by inducing defective meiotic chromosome segregation. The abnormal oocyte karyotype is inherited by embryos, which die in utero at an early stage of development. In addition, embryo death in SCP3-deficient females increases with advancing maternal age. We found that SCP3 is required for chiasmata formation and for the structural integrity of meiotic chromosomes, suggesting that altered chromosomal structure triggers nondisjunction. SCP3 is thus linked to inherited aneuploidy in female germ cells and provides a model system for studying age-dependent degeneration in oocytes.

Abnormal chromosome migration and chromosome aberrations in mouse oocytes during meiosis II in the presence of topoisomerase II inhibitor ICRF-193

Using a mouse parthenogenetic system, effects of ICRF-193, a noncleavable complex-forming topoisomerase II inhibitor, on female meiosis II chromosomes and pronuclear chromosomes were studied. Eggs were exposed to the inhibitor (10 μM) at various times after parthenogenetic stimulation, and chromosomes of them were analyzed at the first cleavage metaphase. When eggs were exposed to the inhibitor during the period from metaphase II to anaphase II, a significant increase in incidences of structural chromosome aberrations (51.1% versus 1.3% in the control) and aneuploidy (30.3% versus 0.7% in the control) was found. Structural chromosome aberrations were observed in 10–20% of eggs following treatments during telophase II, but there was no increased incidence of aneuploidy in treatments during this meiotic stage. When pronuclear eggs at S phase were targeted by the inhibitor, no significant increase in chromosome aberrations was found. Interestingly, when chromatids moved to each pole during anaphase II in the presence of ICRF-193, most of them oriented their centromeres toward the spindle equator as if moving backwards. Moreover, lagging chromatids with the centromeres present were observed in more than 50% of treated eggs. However, chromosomal bridges that resulted from chromosome stickiness did not appear in any egg. These findings indicate that ICRF-193 can induce structural chromosome aberrations and aneuploidy in mouse secondary oocytes in meiotic stage-dependent manner. The induction of aneuploidy is due to disruption of the separation of sister centromeres at anaphase II. There appears to be mechanism(s) other than cleavable complex formation or chromosome stickiness behind the induction of structural chromosome aberrations by ICRF-193.

Ageing and aneuploidy in oocytes.

Correlations between maternal age and Down syndrome have been known already for over half a century (Penrose 1933; Bond and Chandley 1983). Still, the reasons for the dramatic increase in risks for a trisomic conceptus, spontaneous abortion associated with a chromosomally unbalanced embryo and the significantly reduced developmental potential of oocytes and embryos in aged women are unclear. Demographic analysis shows that there still is a trend for delaying childbearing to advanced maternal ages in many industrialized countries. Accordingly, it has been estimated that 25% of conceptions will involve women of 35 years or older in the Netherlands in 2005–2009 (te Velde and Pearson 2002). Many couples attending the infertility clinics are of advanced age. Therefore, it is important to investigate the origin of the maternal age-related decline in fertility associated with aneuploidy in oocytes in order to predict individual risks and, possibly, improve treatment. This contribution reviews briefly the current status of research on the incidence and the origin of aneuploidy in aged oocytes in humans and some experimental animals The observations suggest that prenatal events in oogenesis and recombination patterns influence susceptibility of chromosomes to errors in segregation, but that the depletion of the follicle pool, hormonal homeostasis, the oocyte-specific fragility of cohesion between homologues and permissive cell-cycle regulation at maturation may be important in the reduced quality of aged oocytes, which affects critically the fidelity of chromosome segregation and developmental potential.

MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes

BackgroundAlthough chromosome missegregation during oocyte maturation (OM) is a significant contributor to human morbidity and mortality, very little is known about the causes and mechanisms of aneuploidy. Several investigators have proposed that temporal perturbations during OM predispose oocytes to aberrant chromosome segregation. One approach for testing this proposal is to temporarily inhibit the activity of protein proteolysis during OM. We used the reversible proteasome inhibitor MG-132 to transiently perturb the temporal sequence of events during OM and subsequently analyzed mouse metaphase II (MII) for cytogenetic abnormalities. The transient inhibition of proteasome activity by MG-132 resulted in elevated levels of oocytes containing extra chromatids and chromosomes.ResultsThe transient inhibition of proteasome-mediated proteolysis during OM by MG-132 resulted in dose-response delays during OM and elevated levels of aneuploid MII oocytes. Oocytes exposed in vitro to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p < 0.01) higher levels of MI arrested oocytes and lower frequencies of premature sister chromatid separation in MII oocytes. Furthermore, the proportions of MII oocytes containing single chromatids and extra chromosomes significantly (p < 0.01) increased with MG-132 dosage.ConclusionsThese data suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these findings support a relationship between disturbed proteasomal activity and chromosome segregation, considerable additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms on chromosome segregation during OM.

O-25. Mechanisms of aneuploidies in human oocytes

O-25. Mechanisms of aneuploidies in human oocytes

Oocyte-induced haploidization

This paper describes the technical approach to treatment of age-related oocyte aneuploidy. Although one solution can be oocyte/embryo selection, another is represented by the nuclear transplantation procedure. The efficiency of nuclear transplantation into immature oocytes is described as a way of generating embryos, and the possibility that viable female gametes can be constructed by transfer of diploid somatic cell nuclei into enucleated oocytes. Germinal vesicle (GV)-stage mouse oocytes were collected from unstimulated ovaries and somatic nuclei were obtained from mouse cumulus cells obtained after ovarian stimulation. Spare human GV-stage oocytes were donated from consenting patients undergoing intracytoplasmic sperm injection (ICSI) treatment, and human somatic cells were stromal cells coming from uterine biopsies performed on consenting patients undergoing endometrial cell co-culture. GV ooplasts, prepared by enucleation, were transplanted with either GV or somatic nuclei by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were selected at random for insemination or cytogenetic analysis. GV transplantation was accomplished with an overall efficiency of ˜80 and 70% in the mouse and the human respectively. The maturation rate of 96% (mouse) and 62% (human) following reconstitution was comparable to that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The reconstituted human oocytes were successfully fertilized by ICSI at a rate of 52%. After the transfer of mouse cumulus or human endometrial cell nuclei into enucleated immature oocytes, a polar body was extruded in >40%. In a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, the chromosomes segregated normally at the time of polar body extrusion. The technique of nuclear transplantation itself did not increase the incidence of chromosomal anomalies in the mouse or human, since their oocytes reconstituted with homologous donor GV resumed meiosis to metaphase II and maintained a normal ploidy. In addition, immature mouse ooplasts induced haploidization of transplanted somatic cell nuclei. Although further evaluation of their genetic status is needed, the procedure may offer a realistic way of producing normal oocytes in cases of aged-related infertility. While the procedure is technically similar to cloning, it would generate a unique individual as a result of the contribution of both parental genomes.

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization

Objective: To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body.Design: Prospective case study.Setting: Research laboratories, university hospital.Patient(s): A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI.Main Outcome Measure(s): Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured to metaphase II in vitro.Result(s): Oocyte hyperploidy was diagnosed by M-FISH to be 23, X +15 cht +19 cht +22 cht. The correspond- ing polar body was hypoploid, with a karyotype of 23, X −15 cht −19 cht −22 cht. This was due to unbalanced predivision at meiosis I. Reprobing confirmed karyotype assignments for chromosomes X, 13, 18, and 21.Conclusion(s): The mechanism involved in maternally derived aneuploidy can be defined by using M-FISH to simultaneously karyotype both oocyte and first polar body chromosomes at metaphase II. Multifluor FISH may be useful for investigative studies of maternally derived aneuploidy, which is a major cause of preimplantation waste in natural and assisted reproduction.