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.     

Meiotic and mitotic nondisjunction: lessons from preimplantation genetic diagnosis

Direct testing of the outcome of the first and second meiotic divisions has become possible with the introduction of preimplantation genetic diagnosis (PGD) for aneuploidies. Testing of oocytes by fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies showed that more than half of oocytes from the IVF patients aged 35 years and older had chromosomal abnormalities, which originated from errors in meiosis I or meiosis II, or both: 41.9% of oocytes were aneuploid after meiosis I and 37.3% aneuploid after meiosis II, with 29.1% of these oocytes having both meiosis I and meiosis II errors. As a result, one third of oocytes detected as normal after meiosis I contained the meiosis II errors, and two thirds of those with meiosis II errors were already abnormal following meiosis I. Although the rates of chromosomal abnormalities deriving from meiosis I and II were comparable, meiosis I errors predominantly resulted in extra chromosome (chromatid) material in oocytes, in contrast to a random distribution of extra and missing chromatids after meiosis II. The majority of meiosis I abnormalities were represented by chromatid errors, which seem to be the major source of chromosomal abnormalities in the resulting embryos. Approximately one third of aneuploid oocytes deriving from sequential errors in the first and second meiotic divisions resulted in a balanced karyotype, representing a possible phenomenon of "aneuploidy rescue" during the second meiotic division. However, the majority of the embryos resulting from such oocytes appeared to be abnormal for the same or different chromosome(s), or were mosaic, suggesting a possible predisposition of the resulting embryos to further mitotic errors. Although the origin of a high frequency of mosaicism at the cleavage stage is not sufficiently understood, the mosaic embryos may originate from the chromosomally abnormal oocytes, as a result of a "trisomy rescue" mechanism during the first mitotic divisions, which renders polar body FISH analysis to have important clinical value for reliable pre-selection of aneuploidy-free embryos for transfer.     

Mechanisms and targets involved in maternal and paternal age effects on numerical aneuploidy

Trisomy in the human appears to be predominantly associated with maternal age. The maternal-age effect, however, shows considerable variability across affected chromosomes. Chromosome-specific variation has been reported in the shapes of the maternal-age-effect curves, including very small effects for the large chromosomes (groups A and B), linear increases (chromosome 16), and exponential increases (chromosome 21). There is also variation among chromosomes in whether the segregation errors occur predominantly at maternal meiosis I, meiosis II, and/or postfertilization mitotic divisions. There is also limited epidemiological evidence for a paternal-age effect, which was recently supported by the findings of age-related increases in sperm aneuploidy using fluorescence in situ hybridization methods. The paternal-age effect is considerably smaller than the maternal and is more likely to involve meiotic II errors of the sex chromosomes, whereas the maternal-age effect is more likely to arise from meiotic I errors producing autosomal trisomies. These and other differences suggest that constitutional aneuploidy arises by multiple mechanisms that may affect (1) the nature and timing of an initiating lesion affecting the oocyte or sperm; (2) the cellular physiology at the time of the nondisjunction event at meiosis I, II, or postfertilization; and (3) the selection against specific chromosomal aneuploidies during embryonic development. Multidisciplinary research is needed to understand the maternal- and paternal-age effects on aneuploidy, to (1) identify and characterize the genes that control meiosis, recombination, and segregation; (2) identify the microenvironmental factors around the oocyte and male germ cells that are involved in the age effects; (3) develop a laboratory animal model for the age effects; (4) characterize the role of genetics, physiology, and environmental toxicology for the parental-age effects; and (5) identify cohorts of men and women of differing ages who have been exposed to high doses of candidate aneugens and conduct epidemiological investigations of aneuploidies transmitted to their offspring. © 1996 Wiley-Liss, Inc.     

Fertilization and early embryology: Influence of maternal age on meiotic spindle assembly oocytes from naturally cycling women

To examine the effects of maternal ageing on the meiotic apparatus,we obtained oocytes from naturally cyding women in two age groups,including younger (aged 20–25 years) and older (aged 40–45years) women. Using high- resolution confocal microscopy weobtained a detailed picture of the meiotic spindle and chromosomeplacement during various phases of meiosls. Our data revealedthat the meiotic spindle in older women is frequently abnormal,both with regard to chromosome alignment and the micro- tubulematrix that comprise the meiotic spindle. The spindle in 79%of the oocytes from the older group exhibited abnormal tubulinplacement and one or more chromosomes were displaced from themetaphase plate during the second meiotic division. In contrast,only 17% of the oocytes from the younger age group exhibitedaneuploid conditions. The majority of eggs from this group possesseda well ordered, meiotlc spindle containing chromosomes thatwere fully aligned within a distinct metaphase plate in thespindle. Chromosome management during meiosis is directed bymicrotubule assembly within the spindle. These data suggestthat the regulatory mechanisms responsible for assembly of themeiotic spindle are significantly altered in older women, leadingto the high prevalence of aneuploidy.     

Differential distribution of aneuploidy in human gametes according to their sex.

The cytogenetic study of human gametes is a new and important source of information because most chromosomal abnormalities originate from meiotic disorders. The frequency and type of abnormalities were analysed in both spermatozoa and mature oocytes. A total of 13,975 human sperm chromosome complements and 1897 oocyte chromosome complements were analysed. In the present study, pooled cytogenetic data on human gametes have been examined to determine and compare the distribution of non-disjunctions in male and female gametes. Human spermatozoa are characterized by a significant excess of hypohaploidy and an equal distribution of aneuploidies among all chromosome groups, whereas mature oocytes display an equal ratio of hypohaploidy to hyperhaploidy and a high variability in the distribution of non-disjunction: in particular, there is a significant over-representation of aneuploidies in both D and G chromosome groups. This indicates that non-disjunction is not a random event in female meiosis and, consequently, that there are differences in the meiotic process between the sexes. Meiotic and environmental factors which could explain the non-random malsegregation of chromosomes in female meiosis are discussed. The role of maternal age as a cause of aneuploidy is questioned.     

The high incidence of meiotic errors increases with decreased sperm count in severe male factor infertilities

BACKGROUND:The high frequency of aneuploidy sperm raises concerns that there may be an increased incidence of aneuploid offspring in ICSI programmes. In order to assess the role that chromosome complement plays in normal and abnormal fertility, detailed molecular cytogenetic studies must be done on sperm samples from men with normal and abnormal fertility. METHODS: To understand more clearly the cytogenetic make-up of sperm from oligoasthenoteratozoospermic (OAT) patients, multi-colour fluorescence in situ hybridization was used to determine numerical chromosome abnormalities. RESULTS: Increased aneuploidy frequencies for chromosomes 13, 18, 21, X and Y were detected in sperm from OAT patients. The frequencies of diploidy also increased. There were no differences in non-disjunction at meiosis I compared to meiosis II. Sperm count inversely correlated with the frequencies of diploidy, aneuploidies for chromosomes 13 and 21 in OAT patients. Twenty-two cycles of ICSI and 18 embryo transfers were performed in 20 couples. Only three cases achieved successful pregnancies. CONCLUSIONS: A higher incidence of meiotic errors and lower sperm counts was found in sperm from OAT patients.     

Double non-disjunction in maternal meiosis II giving rise to a fetus with 48,XXX,+21.

We describe a prenatally detected case of double trisomy involving chromosome 21 and the X chromosome (48,XXX,+21) along with determination of the segregation errors responsible for the double aneuploidy. The patient was ascertained as a result of an abnormal maternal serum analyte screen showing an increased risk for fetal Down's syndrome. Following determination of the abnormal karyotype, pregnancy termination was elected. Microsatellite polymorphisms and cytogenetic heteromorphisms were used to determine that both aneuploidies arose as a result of non-disjunction in maternal meiosis II. These results support hypotheses that a segregation defect at a cellular level may cause non-disjunction involving more than one chromosome.     

Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization

Numerical chromosome errors are known to be common in early human embryos and probably make a significant contribution to early pregnancy loss and implantation failure in IVF patients. Over recent years fluorescent in situ hybridization (FISH) has been used to document embryonic aneuploidies. Many IVF laboratories perform preimplantation genetic diagnosis (PGD) with FISH to select embryos that are free from some aneuploidies in an attempt to improve implantation, pregnancy and live birth rates in particular categories of IVF patients. The usefulness of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. Complete karyotyping at the single cell level can now be achieved by comparative genomic hybridization (CGH). CGH enables not only enumeration of all chromosomes but gives a more complete picture of the entire length of each chromosome and has demonstrated that chromosomal breakages and partial aneuploidies exist in embryos. CGH has provided invaluable information about the extent of mosaicism and aneuploidy of all chromosomes in early human conceptuses. CGH has been applied to clinical PGD and has resulted in the birth of healthy babies from embryos whose full karyotype was determined in the preimplantation phase.     

Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization

Chromosomal aneuploidies contribute considerably to the lowpregnancy rate in in-vitro fertilization (IVF). The objectiveof this experimental work was to explore the possibility ofdetecting common aneuploidies in oocytes by polar body sampling.The study included 45 infertile patients of advanced maternalage participating in an IVF programme. The first polar bodywas removed prior to fertilization or both the first and secondpolar bodies were removed after fertilization and studied byfluorescent in-situ hybridization (FISH) using chromosome-specificprobes for chromosomes X, 18 and/or 13/21. Of 155 oocytes withFISH results, 36 demonstrated chromosomal abnormalities. Of119 oocytes predicted to be free from aneuploidy of chromosomesX, 18 and/or 13/21, 72 were normally fertilized, cleaved andtransferred in 23 treatment cycles, which resulted in two healthydeliveries and three ongoing pregnancies confirmed to be unaffectedby chorionic villous sampling. The method may appear usefulfor the detection of oocytes with common chromosomal aneuploidiesin IVF patients of advanced maternal age. chromosomal aneuploidies/fluorescent in-situ hybridization/human first and second polar bodies/pre-conception/preimplantation genetic diagnosis     

Diagnosing and preventing inherited disease: Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization

Chromosomal aneuploidies contribute considerably to the lowpregnancy rate in in-vitro fertilization (IVF). The objectiveof this experimental work was to explore the possibility ofdetecting common aneuploidies in oocytes by polar body sampling.The study included 45 infertile patients of advanced maternalage participating in an IVF programme. The first polar bodywas removed prior to fertilization or both the first and secondpolar bodies were removed after fertilization and studied byfluorescent in-situ hybridization (FISH) using chromosome-specificprobes for chromosomes X, 18 and/or 13/21. Of 155 oocytes withFISH results, 36 demonstrated chromosomal abnormalities. Of119 oocytes predicted to be free from aneuploidy of chromosomesX, 18 and/or 13/21, 72 were normally fertilized, cleaved andtransferred in 23 treatment cycles, which resulted in two healthydeliveries and three ongoing pregnancies confirmed to be unaffectedby chorionic villous sampling. The method may appear usefulfor the detection of oocytes with common chromosomal aneuploidiesin IVF patients of advanced maternal age.     

Aneuploidy 12 in a Robertsonian (13;14) carrier: Case report

In translocation carriers, the presence of aneuploidy for the chromosomes unrelated to the rearrangement may lead to an additional risk of abnormal pregnancy or implantation failure. Consequently, it may be important to analyse not only the chromosomes involved in the rearrangement but also the rest of chromosomes. We combined spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to karyotype one unfertilized oocyte and its first polar body (1PB) from a Robertsonian translocation carrier t(13;14) aged 29 years who was undergoing IVF and preimplantation genetic diagnosis (PGD) for translocations and aneuploidy screening. Two out of four embryos were aneuploid, as a result of an adjacent segregation. The unfertilized oocyte had a normal/ balanced constitution of the chromosomes involved in the reorganization. However, this 1PB-metaphase II doublet was aneuploid for chromosome 12, the oocyte being hyperhaploid (24, X, +12) and its 1PB hypohaploid (22, X, -12). The application of CGH for the study of Robertsonian translocations of maternal origin will be useful to study imbalances of the chromosomes involved in the rearrangement, as well as alterations in the copy number of any other chromosome. The combination of PGD for translocations with aneuploidy screening could help to reduce the replacement of chromosomally abnormal embryos.     

The contradictory information on the distribution of non-disjunction and pre-division in female gametes

Valuable information on the cytogenetic constitution of female gametes has been deduced from the direct, so-called conventional analysis of oocytes remaining unfertilized in programmes of assisted reproduction. Additional, indirect conclusions have become possible by PGD of the polar bodies. Both techniques provided evidence for the co-existence of two aneuploidy-causing mechanisms during first maternal meiosis; non-disjunction (ND) of bivalents results in the loss or gain of whole chromosomes in metaphase II complements, whereas a precocious division (pre-division, PD) of univalents leads to the loss or gain of single chromatids. As to the distribution of ND and PD, however, direct oocyte chromosome studies and PGD tell surprisingly different stories. Moreover, first and second polar body analyses contradict the data derived from DNA polymorphism studies concerning the distribution of first and second meiotic division errors. An increased awareness of these problems appears necessary because important decisions are made on the basis of PGD results.     

Parental age-related aneuploidy in human germ cells and offspring: A story of past and present

Parental age is the most important aetiological factor in trisomy formation in humans. Cytogenetic studies on germ cells reviewed here imply that (i) 2–4% sperm are aneuploid, and 8.6% oocytes from IVF are hyperploid, (ii) a paternal age effect may exist, and (iii) oocytes of aged women contain precociously separated chromatids in metaphase II. Trisomy data suggest that most aneuploidy is generated during meiosis 1 of oogenesis and is maternal age-dependent. Trisomy 18 is unique, originating mostly from maternal meiosis II errors. The extra gonosome in 47, XXY derives mostly from a paternal meiosis I error. Trisomy of individual chromosomes may remain low, linearly rise, or exponentially increase with advanced maternal age. Maternal age related trisomies involve achiasmatic and normochiasmate chromosomes, and chromosomes with disturbed recombination and distally located chiasmata. Hypotheses on the origin of the maternal age-effect are critically reviewed. One model is presented that relates to altered cell cycle and protein phosphorylation in oocytes of aged mammals and accounts for most of the observed data in humans and in experimental studies. Aneuploidy may thus involve a predetermined component but is possibly also influenced by extrinsic factors reducing oocyte quality or depleting the oocyte pool precociously. Areas of future research are proposed to elucidate (i) the significance of early disturbances in the prenatal ovary, (ii) parameters diminishing the quality of oocytes in dictyate stage, and (iii) mechanisms enabling oocytes to process all chromosomal configurations successfully during later stages of oogenesis. Studies with newly developed and existing animal models appear indispensable to identify exposures affecting chromosome disjunction during meiosis, especially in the aging female. © 1996 Wiley-Liss, Inc.     

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.      

Variable aneuploidy mechanisms in embryos from couples with poor reproductive histories undergoing preimplantation genetic screening

BACKGROUND: Preimplantation genetic screening (PGS) is used to determine the chromosome status of human embryos from patients with advanced maternal age (AMA), recurrent miscarriage (RM) or repeated implantation failure (RIF). METHODS: Embryos from 47 such couples were investigated for chromosomes 13, 15, 16, 18, 21 and 22 using fluorescence in situ hybridization with two rounds of hybridization. The investigation included parental lymphocyte work-up, the screening of blastomeres on day 3 and full follow-up on day 5/6 of untransferred embryos. RESULTS: The outcome of 60 PGS cycles is described, in which 523 embryos were biopsied; 91% gave results, of which 18% were diploid for all the chromosomes tested and 82% were abnormal. The pregnancy rate per cycle that reached the biopsy stage was 27%, and 30% per embryo transfer. Satisfactory follow-up was obtained from 353 embryos; all those diagnosed as abnormal were confirmed as such, although two false-positives were detected in relation to specific chromosome abnormalities. Meiotic errors were identified in 16% of embryos. Between the RM, AMA and RIF groups, there was a significant difference in the distribution of embryos that were uniformly abnormal and of those with meiotic errors; with an almost 3-fold increase in meiotic errors in the first two groups compared with the RIF group. CONCLUSIONS: This complete investigation has identified significant differences between referral groups concerning the origin of aneuploidy in their embryos.     

Origin of nondisjunction in trisomy 21 syndrome: all studies compiled, parental age analysis, and international comparisons

The Q-band polymorphism of chromosome 21 permits assignment of the origin of meiotic nondisjunction by parent and meiotic division in a certain proportion of cases. We have compiled all reports through 1982 (including earlier studies using structural abnormality) and have shown that maternal origin accounts for 80% and paternal origin for 20% of trisomic cases. The ratio of first:second meiotic errors among the maternal cases was 80:20 and 60:40 among the paternal cases. Considering maternal and paternal first and second meiotic errors, we showed no differences of either mean maternal or paternal age, though mean maternal age is about 5 yr higher than that of the general population. Comparison of results of six studies from five countries showed similar frequencies by parent and meiotic division with the possible exception of one study from the northeastern United States. The causative role of simply advancing maternal age in nondisjunction is questioned.     

Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure

Using comparative genomic hybridization, we have detected chromosome abnormality in 76/126 (60%) single blastomeres biopsied prior to implantation from embryos from 20 women with repeated implantation failure following IVF. The abnormalities detected included aneuploidy for one or two chromosomes [32/126 (25%)] and complex chromosomal abnormality [37/126 (29%)]. Most of the chromosomes involved in single aneuploidy were those commonly found in live births or spontaneously aborted fetuses, whereas a greater range of chromosomes were involved in double aneuploidy. In blastomeres with complex abnormality, random and extensive loss and gain of all the chromosomes was observed. Further blastomeres from 25 embryos with single or double aneuploidy and 11 embryos with complex abnormality were analysed following embryo disaggregation. The specific abnormality was confirmed in the majority of cases and in some cases could be assigned as errors in meiotic or mitotic segregation. Complex abnormalities, suggestive of errors in cell cycle regulation, were present in a slightly higher proportion of these embryos than were seen in our previously studied cohort of surplus embryos. The disruption of the normal sequence of chromosome replication and segregation in early human embryos, caused either by maternal cytoplasmic factors or mutations in cell cycle control genes, may be a common cause of repeated implantation failure.     

Loss of the SHOX gene associated with Leri-Weill dyschondrosteosis in a 45,X male

A male patient is reported with a 45,X karyotype and Leri-Weill dyschondrosteosis (LWD). FISH analysis with SHOX and SRY gene probes was carried out. One copy of both SHOX and SRY was detected in interphase nuclei, clarifying the origin of LWD and the male phenotype. Molecular results suggested that the 45,X karyotype arose through two independent events. The first occurred at paternal meiosis leading to an unequal crossing over between the short arms of the X and Y chromosomes. As a consequence, the SRY gene was translocated onto Xp, thereby explaining the male phenotype of the patient. The second event probably occurred at maternal meiosis or at the early stages of the zygote resulting in the loss of the maternal X chromosome.     

Cytogenetic analysis of unfertilized oocytes retrieved after treatment with the LHRH analogue, buserelin

Chromosome analysis is reported on 155 oocytes from an in-vitro fertilization (IVF) programme in which the LHRH analogue, buserelin, was used in the superovulation regime. Seventy-one oocytes had the normal number of metaphase II chromosomes. Hyperhaploidy was apparent in four cases only; doubling this figure to include the reciprocally formed hypohaploid oocytes gave an aneuploidy rate due to non-disjunction of 10%. This is close to the figure for naturally occurring aneuploidy which may be calculated from data on recognized conceptions at a comparable maternal age. Taken together with the suggestion of a maternal age effect in our series these data suggest that follicular stimulation regimes which precede IVF do not necessarily add to the naturally high aneuploidy rate of the human species. Thirteen oocytes had failed to form the first polar body and the presence of diploid mitotic or sperm chromosomes provided evidence of fertilization and arrested development in 15. These figures for chromosomal anomalies following buserelin treatment are not significantly different from those obtained from comparable surveys following clomiphene citrate stimulation, providing no evidence that the improved pregnancy rate with buserelin is due to chromosomal factors.     

Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age

Mammalian oocytes begin meiosis in the fetal ovary, but only complete it when fertilized in the adult reproductive tract. This review examines the cell biology of this protracted process: from entry of primordial germ cells into meiosis to conception. The defining feature of meiosis is two consecutive cell divisions (meiosis I and II) and two cell cycle arrests: at the germinal vesicle (GV), dictyate stage of prophase I and at metaphase II. These arrests are spanned by three key events, the focus of this review: (i) passage from mitosis to GV arrest during fetal life, regulated by retinoic acid; (ii) passage through meiosis I and (iii) completion of meiosis II following fertilization, both meiotic divisions being regulated by cyclin-dependent kinase (CDK1) activity. Meiosis I in human oocytes is associated with an age-related high rate of chromosomal mis-segregation, such as trisomy 21 (Down's syndrome), resulting in aneuploid conceptuses. Although aneuploidy is likely to be multifactorial, oocytes from older women may be predisposed to be becoming aneuploid as a consequence of an age-long decline in the cohesive ties holding chromosomes together. Such loss goes undetected by the oocyte during meiosis I either because its ability to respond and block division also deteriorates with age, or as a consequence of being inherently unable to respond to the types of segregation defects induced by cohesion loss.