Chromosome Aberrations In Embryos Research Articles

THE LANDSCAPE OF CHROMOSOMAL ABERRATIONS IN 10,000 EMBRYOS DERIVED FROM FRESH DONOR OOCYTES

Information on chromosomal aberration rates in embryos derived from a young population, such as egg donors, is limited. PGT-A is the best predictive tool for embryo selection. It is most often offered to patients with advanced age, but with contradicting recommendations in younger patients. The objective was to evaluate the type and frequency of chromosomal aberrations in embryos derived from fresh young donor oocytes detected at NGS resolution of 10Mb and >30% mosaicism.

P-564 Advanced paternal age is associated with higher rate of segmental chromosomal aberrations in preimplantation embryos

Abstract Study question Is there a correlation between paternal age and the frequency of chromosomal aberrations in embryos created by in vitro fertilization (IVF)? Summary answer There is an association between paternal age and segmental aberration rates in embryos derived from young oocyte providers after adjusting for age,sperm,and IVF-cycle characteristics. What is known already Paternal reproductive aging(APA) is less understood than maternal reproductive aging(AMA). Embryo aneuploidy presents one of the most important determinants of transfer cycle success. The association of AMA with increased rate of aneuploidy is well established. The systematic review and meta-analysis published by our group did not identify an association of APA with aneuploidy, based on ∼10,000 pooled embryos, however, the power to detect subchromosomal aberrations was limited by the sensitivity of the PGT-A platform used by each center. Our aim was to study the impact of APA on chromosomal aberrations in a large sample size analyzed by high resolution PGT-A. Study design, size, duration This was a retrospective cohort study performed at the CReATe Fertility Centre Genetics Laboratory, Toronto, Canada, between January 2019-December 2021. A total of 17731 blastocysts from 3506 cycles (2502 patients) were analyzed using high resolution NGS PGT-A (10Mb resolution; 30%-70% reported as mosaicism). Clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: ≤33years and ≥34years. Each group was divided into four sub-groups by paternal age: Group A 20-29(n = 484); GroupB 30-39(n = 9704); GroupC 40-49(n = 6341); GroupD≥50(n = 1202). Multiple logistic/ordinal regressions were conducted to evaluate euploidy, mosaicism and aneuploidy rates between age groups, as appropriate. Each was further sub-analyzed by type of abnormality A-single-whole-chromosome, S-segmental, C-complex (≥2 full-chr-aberrations). Segmental aberrations were divided into subgroups of S + (segmental gain), S-(segmental loss) and CS(complex segmental). Main results and the role of chance High resolution NGS PGT-A analysis of 17731 embryos showed 54.9% were euploid, 31.5% were aneuploid and 13.6% mosaic. In the oocyte provider’s age of ≤ 33 group, significant differences between paternal age group A vs. B, C and D were found in euploidy rates (68.8%, 62.5%, 61.7%, 59.0% respectively; p = 0.00559). There were no significant differences in aneuploidy rates (19.1%, 22.7%, 22.5%, 24.6% respectively; p = 0.07184) or mosaicism rates (12.1%, 14.8%, 15.8%, 16.3%; respectively, p = 0.23754). In the oocyte provider’s age of ≥ 34 group, decreasing euploidy rates and increasing aneuploidy rates were observed between all paternal age groups (p = 0.00001 and p < 0.00001,respectively), and no significant differences in mosaicism rates (14%, 12%, 10.2%, 11.6% respectively) reflecting the maternal age effect on euploidy/aneuploidy. In the oocyte provider’s age of ≤ 33 group, paternal group A (2.3%) had significantly lower segmental (S) rates compared to groups B (4.3%, p = 0.027599), C (3.3%, p = 0.039924) and D (4.6%, p = 0.018478). In addition, segmental losses (S-) were lower in group A (1.3%) compared to groups B (3.4%, p = 0.020431), C (3.1%, p = 0.043166) and D (3.7%, p = 0.016385). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The results of this study provide important information to assist fertility practitioners when providing pre-conception counseling to patients, particularly for couples in which the male partner belongs to an older age group. Trial registration number na

Is there an association between paternal age and aneuploidy? Evidence from young donor oocyte-derived embryos: a systematic review and individual patient data meta-analysis.

Delayed parenthood, by both women and men, has become more common in developed countries. The adverse effect of advanced maternal age on embryo aneuploidy and reproductive outcomes is well known. However, whether there is an association between paternal age (PA) and embryonic chromosomal aberrations remains controversial. Oocyte donation (OD) is often utilized to minimize maternal age effects on oocyte and embryo aneuploidy, thus providing an optimal model to assess the effect of PA. Several studies have revealed a higher than expected rate of aneuploidy in embryos derived from young oocyte donors, which warrants examination as to whether this may be attributed to advanced PA (APA). The objective of this systematic review and individual patient data (IPD) meta-analysis is to evaluate existing evidence regarding an association between PA and chromosomal aberrations in an OD model. This review was conducted according to PRISMA guidelines for systematic reviews and meta-analyses. Medline, Embase and Cochrane databases were searched from inception through March 2020 using the (MeSH) terms: chromosome aberrations, preimplantation genetic screening and IVF. Original research articles, reporting on the types and/or frequency of chromosomal aberrations in embryos derived from donor oocytes, including data regarding PA, were included. Studies reporting results of IVF cycles using only autologous oocytes were excluded. Quality appraisal of included studies was conducted independently by two reviewers using a modified Newcastle-Ottawa Assessment Scale. A one-stage IPD meta-analysis was performed to evaluate whether an association exists between PA and aneuploidy. Meta-analysis was performed using a generalized linear mixed model to account for clustering of embryos within patients and clustering of patients within studies. The search identified 13 032 references, independently screened by 2 reviewers, yielding 6 studies encompassing a total of 2637 IVF-OD cycles (n = 20 024 embryos). Two 'low' quality studies using FISH to screen 12 chromosomes on Day 3 embryos (n = 649) reported higher total aneuploidy rates and specifically higher rates of trisomy 21, 18 and 13 in men ≥50 years. One 'moderate' and three 'high' quality studies, which used 24-chromosome screening, found no association between PA and aneuploidy in Day 5/6 embryos (n = 12 559). The IPD meta-analysis, which included three 'high' quality studies (n = 10 830 Day 5/6 embryos), found no significant effect of PA on the rate of aneuploidy (odds ratio (OR) 0.97 per decade of age, 95% CI 0.91-1.03), which was robust to sensitivity analyses. There was no association between PA and individual chromosome aneuploidy or segmental aberrations, including for chromosomes X and Y (OR 1.06 per decade of age, 95% CI 0.92-1.21). Monosomy was most frequent for chromosome 16 (217/10802, 2.01%, 95% CI 1.76-2.29%) and trisomy was also most frequent for chromosome 16 (194/10802, 1.80%, 95% CI 1.56-2.06%). We conclude, based on the available evidence, that APA is not associated with higher rates of aneuploidy in embryos derived from OD. These results will help fertility practitioners when providing preconception counselling, particularly to older men who desire to have a child.

19. PGT-A: WHEN IT IS BETTER NOT TO KNOW

Introduction PGT-A was implemented to select euploid embryos and thus, to improve IVF-outcome in terms of higher live birth rate (LBR), lower miscarriage rate or shorter time-to-pregnancy. With the implementation of refined techniques such as Next Generation Sequencing (NGS) the resolution to detect (small) chromosomal aberrations in embryos increased. This holds true for as chromosomal mosaicism as well as for segmental aberrations, and consequently the amount of embryos tested as euploid decreases. This observation highlights the complex nature of genetic (in)stability during early embryogenesis and reveals the substantial shortcomings of PGT-A. The early genetic plasticity counteracts with the primary goal to improve LBR. The aim of the study was to determine when PGT-A can be recommended considering the drop-out rate starting from non-biopsable embryos, amplification failure and drop-out due to testing as non-euploid. Patients, material & methods This retrospective single-center study (2016-2018) includes 173 hormonal stimulation cycles (71 patients). PGT-A was performed either due to three unsuccessful embryo transfers or due to three miscarriages in the patient's history. Patients with tested structural chromosomal aberrations as well as donor cycles were excluded. Blastocyst culture was performed in single step culture medium in the Embryoscope. TE-biopsy was performed on day 5-7. WGA was performed either by PicoPlex (Rubicon Genomics) or by SurPlex (BlueGenome/ Illumina). NGS was processed using VeriSeq platform with data analysis by BlueFuse Multi (Illumina). Results A total of 1,751 MII oocytes resulted in 752 blastocysts. TE-biopsy of at least one blastocyst was performed in 157 cycles (68 patients). This resulted in 609 biopsied blastocysts of which 57 showed either failure of amplification or defective sequencing. 156 (25.6%) samples showed either whole chromosome aneuploidies or uniform segmental chromosomal aberrations. Further, 117 (19.2%) biopsies revealed either chromosomal or segmental mosaicism and, 140 (23.0%) were tested with a combination of uniform and mosaic aberrations. Finally, 139 (22.8%) samples were tested as chormosomally normal. This results in mean 0.8 blastocysts/cycle tested as known euploid compared to originally 4.5 blastocysts/cycle. Conclusions High embryo losses due to non-biopsable blastocysts, amplification failure and non-interpretable results are well known but often neglected. Additionally, while NGS allows high resolution for small structural or numerical chromosomal aberrations as mosaic, the number of transferable blastocysts decreases enormous too. Chromosomal mosaicism (euploid/aneuploid) is a major problem of PGT-A, additional problems arises from segmental chromosomal aberrations that can be present as mosaic as well. When PGT-A is recommended IVF-patients, physicians have to inform about high costs for patients, the increased number of IVF-cycles needed and the high cancellation rates. When estimating a 60% baby-take home rate after PGT-A the same number of babies would be born starting with 752 untested blastocysts considering an only 11% baby-take home rate (83.4 vs. 82.7 babies). This comparison shows that counselling for PGT-A due to RIF or RM has to be well prepared.

Association between chromosomal translocation imbalances and aneuploidies in PGT-SR in reciprocal translocation carriers

Introduction Reciprocal translocation carriers are known to be at high risk of conceiving genetically unbalanced embryos, which may result in miscarriage or an affected neonate. Preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR), a method for embryonic chromosomal analysis, can detect not only translocation chromosome imbalance but also aneuploidy via comprehensive chromosomal analysis with whole-genome amplification (WGA), thereby potentially decreasing miscarriage rate. In this study, we aimed to investigate the relationship between chromosomal abnormalities derived from translocation chromosomes and aneuploidies and the frequency of chromosomal abnormalities among stimulation cycles in reciprocal translocation carriers receiving multiple PGT-SR cycles. Material and Method Between 2013 and 2017, we assessed 20 cycles of 8 couples (reciprocal translocation carriers in 5 females and 3 males; mean women's age, 35.1 years) who received 2 or more PGT-SR cycles at our clinic.Ovarian stimulation was primarily controlled, and when chromosomally normal embryos could not be obtained, a different protocol for ovarian stimulation was adapted. All oocytes were fertilized via intracytoplasmic sperm injection (ICSI). All embryos were cultured to the blastocyst stage. Approximately 5-10 trophectoderm (TE) cells were subjected to a biopsy using a laser system. Chromosomal analysis was performed using the microarray-based comparative genomic hybridization method or next-generation sequencing with whole-genome amplification. Result In all 20 cycles, the number of oocytes retrieved was 432 and the number of blastocysts subjected to biopsy was 90. Among 88 analyzable blastocysts, 19 had normal chromosomes (21.6%) and 69 had abnormal chromosomes (78.4%). Fifty-six embryos (81.2%) contained chromosomal imbalances derived from reciprocal translocation chromosomes and 37 embryos (53.6%) had aneuploidy, 24 embryos (34.8%) had both types of abnormalities. The incidence of chromosomal translocation-derived chromosomal imbalance was 60.0% (12/20) for male carriers and 64.7% (44/68) for female carriers, with no significant difference.The incidence of chromosomally normal embryos per cycle was 0–67%. Normal embryos were not obtained in 9 cycles (45%); however, they were obtained at different stimulation cycles. In 9 PGT-SR cycles with three or more embryos of female carriers subjected to biopsy, there was no correlation between the incidence of the abnormality due to chromosome translocation and aneuploidy. Conclusion There is no association between the incidence of translocation chromosome imbalance and aneuploidy. However, approximately 80% of embryos with chromosomal translocation-derived chromosomal imbalance include aneuploidy. The incidence of chromosomal aberrations in embryos has a large difference among the PGT-SR cycles even in the same carrier. In translocation carriers without normal embryos, performing different ovarian stimulation methods enhances the possibility of obtaining normal embryos.

Risk of chromosomal aberration in spermatozoa during intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has become critical for the treatment of severe male infertility. The principal feature of ICSI is the direct injection of spermatozoon into an oocyte, which facilitates the production of fertilized embryos regardless of semen characteristics, such as sperm concentration and motility. However, the chromosomal integrity of ICSI zygotes is degraded compared to that of zygotes obtained via in vitro fertilization. This chromosomal damage may occur due to the injection of non-capacitated, acrosome-intact spermatozoa, which never enter the oocytes under natural fertilization. Furthermore, it is possible that the in vitro incubation and pre-treatment of spermatozoa during ICSI results in DNA damage. Chromosomal aberrations in embryos induce early pregnancy losses. However, these issues may be overcome by embryo production using gametes with guaranteed chromosomal integrity. Because conventional chromosome analysis requires fixing cells to obtain the chromosome spreads, embryos cannot be produced using the nucleus that has been analyzed. On the other hand, genome cloning using androgenic or gynogenic embryos provides an additional nucleus for chromosome analysis following embryo production. Thus, this review aims to highlight the hazardous nature of chromosomal aberrations in sperm during ICSI and to introduce a method for the prezygotic examination for chromosomal aberrations.

Shallow whole genome sequencing is well suited for the detection of chromosomal aberrations in human blastocysts

To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation genetic diagnosis (PGD) in translocation carriers. Study of diagnostic accuracy. University hospital. Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. Trophectoderm biopsy was performed on 47 blastocysts. In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a ∼ 4.5 Mb deletion/duplication. This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.

Incidence of Chromosomal Aberrations in Embryos From a Fertile Group of Patients

Preimplantation Genetic Diagnosis (PGD) has been used primarily for the detection of chromosomal aberrations in embryos of patients with recurrent miscarriages or failed implantation. Unlike other patient populations where PGD has been applied, who suffer either from certain degree of infertility and/or genetic defects, our PGD program primarily treats patients for family balancing, as well as aneuploidy screening. The aim of the study was to investigate the incidence of chromosomal abnormalities in this group of fertile patients.

Paternally transmitted chromosomal aberrations in mouse zygotes determine their embryonic fate.

The developmental consequences of chromosomal aberrations in embryos include spontaneous abortions, morphological defects, inborn abnormalities, and genetic/chromosomal diseases. Six germ-cell mutagens with different modes of action and spermatogenic stage sensitivities were used to investigate the relationship between the types of cytogenetic damage in zygotes with their subsequent risk of postimplantation death and of birth as a translocation carrier. Independent of the mutagen used, over 98% of paternally transmitted aberrations were chromosome type, rather than chromatid type, indicating that they were formed during the period between exposure of male germ cells and initiation of the first S phase after fertilization. There were consistent one-to-one agreements between the proportions of a) zygotes with unstable aberrations and the frequencies of dead embryos after implantation (slope = 0.87, confidence interval [CI]: 0.74, 1.16) and b) zygotes with reciprocal translocations and the frequency of translocation carriers at birth (slope = 0.74, CI: 0.48, 2.11). These findings suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development and that development appears to proceed to implantation regardless of the presence of chromosomal abnormalities. Our findings support the hypothesis that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already set by the end of G1 of the first cell cycle of development.

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Mutagenic and Teratogenic Effects of Indomethacin and Cyclosporine-A on Dams and Fetuses of Mice.

Eighty pregnant female mice were alloted among 16 groups. The animals were given intraperitoneal injection equalized to the therapeutic doses used for human. Indomethacin was given (25, 75 mg/kg b.wt.) and cyclosporine-A was given (5, 10, 15 mg/kg b.wt.). They induced significant increase in fetal resorption and significant decrease in fetal body weight. Also, reduction in the sizes of the skeletons of embryos was observed. Dosage of 150 mg/kg b.wt. of indomethacin was the lethal dose to dams. Various chromosomal aberrations in maternal bone marrow cells and embryos cells and mitotic activity were recorded, quantitated and statistically analyzed. Indomethacin (75 mg/kg) induced more chromosomal aberrations in both pregnant females as well as fetuses than the lower dose (25 mg/kg). Cyclosporine-A (15 mg/kg b.wt.) induced an increase in chromosomal aberrations in bone marrow cells of female mice than lower dose (5, 10 mg/kg b.wt.), while no significant differences were observed between the (5, 10 mg/kg) doses. Also, cyclosporine-A (15 mg/kg) showed highly significant increase in chromosomal aberrations in embryos than medium (10 mg/kg) or lower doses (5 mg/kg).

Analysis of colchicine-induced chromosomal aberrations in embryos obtained from xo mice

A high incidence of chromosomal anomalies has been observed in the offspring of our XO mouse colony. However, the reason(s) are unknown. We hypothesized that XO dams might be more susceptible to certain chromosomal mutagens. Therefore, we assessed whether the oocytes of XO mice are more susceptible to colchicine. Pregnant XO and XX mice were intraperitoneally (i.p.) injected with colchicine, 0.2 mg/kg, 6 h after mating (between resumption of meiosis II and extrusion of the second polar body). Contrary to our expectation, the incidence of chromosomal anomalies induced by colchicine exposure did not differ between XX and XO dams. These findings suggest that the chromosomal stability of the oocytes from XO mice may not be affected by colchicine exposure at the stage of extrusion of the second polar body. The effects of chromosomal imbalance inherent in XO mice on chemical susceptibilities should be further investigated.

Molecular and cellular pathology: summary

Introduction. The aim of the molecular and cellular pathology group in the Bremerhaven Workshop was to test for evidence of biological changes which were indicative of exposure to toxic chemicals, as well as to their adverse effects. Much of the study focused on the liver of the flatfish dab Llmanda limanda. The liver was chosen as it is an integrator of many functions including detoxication/activation of toxic chemicals, digestion and storage, excretion and synthesis of the egg yolk protein vitellogenin. There is also a considerable body of literature on pollutant chemical impact on the cellular pathology of fish liver to aid interpretation. The approach was based on the detection of molecular and cellular changes resulting from chemical contamination. The identification of early onset changes, which may ultimately lead to overt disease is a key requirement in assessment of the effects of environmental pollution. Diseased fish are likely to be rapidly eliminated from the population, hence assessment based on such fish may prove difficult, and even if samples are available, higher level complications resulting from the primary lesion will be a confounding factor. In fact, it is only through the mechanistic linking of changes in molecular and cellular processes with the later pathological endpoints that it will be possible to establish causal relationships. An additional component of the investigation examined developmental abnormalities and chromosomal aberrations in embryos of dab sampled along the transect. An integrated perspective. The results of this investigation of dab in the North Sea show a considerable degree of internal consistency, as well as clearly following the trend of declining levels of chemical contaminants along the transect towards the central North Sea. A pattern emerges from the data showing lysosomal, cellular, tissue and embryonic damage of the type expected due to the toxic action of xenobiotics. The additional evidence of proliferation of the smooth endoplasmic reticulum in hepatocytes, together with an increase in the activity of the inducible detoxicatiodactivation enzyme EROD, further supports the premise that the fish have been exposed to inducerxenobiotics, which is consistent with the analytical chemical data (Cofino et al. 1992). Proliferation of smooth endoplasmic reticulum (SER) and induction of EROD also provide a reasonable mechanistic explanation for the enhanced generation of superoxide radicals, through futile cycling, at the most contaminated sites, while radical attack on lysosomes is probably a contributory factor to the clear evldence for lysosomal membrane injury and consequent dysfunction. The presence of phospholipidosis (accumulation of phospholipid membrane components in lysosomes) in severely damaged livers is also consistent with autophagy of chemically damaged proliferated endoplasmic reticulum and failure of the functionally impaired lysosomes to degrade the ingested membranes, which results in the characteristic pathological presentation of this lysosomal storage disease condition. The presence of foci of altered cells at the most contaminated sites needs to be interpreted carefully. Comparison with the data from other species lends support to the conclusion that the high prevalence of foci is indicative of exposure to xenobiotic pollutants. The relationship of foci to neoplastic change in dab cannot at the present state of our knowledge be determined and requires further experimental investigation. It is, perhaps, worth commenting that neoplastic change in itself is of little significance in ecological terms unless the incidence in the population is extremely high. The value of pursuing the 'neoplastic pathway' is that this type of disease is probably indicative of exposure to carcinogenic xenobiotics, although

Induction and transmission of chemically induced chromosome aberrations in female germ cells.

Female mice were dosed with a number of chemical clastogens. The dosed females were then mated 28.5, 6.5, and 0.5 days after dosing, corresponding to various developmental stages of oogenesis (ie, early pre-antral to late antral stages). One-cell embryos derived from these mating were then isolated and analysed for structural and/or numerical chromosome aberrations. Chromosome damage, either structural or numerical, was induced only in oocytes at the late antral stages of oogenesis (i.e. when females were dosed 0.5 days prior to mating). There were few chromosome aberrations in embryos obtained from females dosed 6.5 or 28.5 days prior to mating. The results show that the analysis of one-cell embryos derived from the germ cells of dosed females is a potentially useful test system for the identification of chemically induced structural and numerical chromosome aberrations. However, the technically demanding methods involved in the preparation of these one-cell embryos prevent their use for routine screening purposes.

Potentiating effect of caffeine on embryotoxicity of cyclophosphamide treatment in vivo during the preimplantation period.

Since it is unknown if chemicals which are generally safe in pregnancy can potentiate the embryotoxicity of cytotoxic drugs before implantation, the combined effects of cyclophosphamide (CPA) and caffeine (CF) were studied in the mouse (day 2) with CPA at 20 mg/kg, which induces a 18% resorption rate, and CF at 100 mg/kg, which does not increase embryolethality. The embryotoxicity of CPA did not increase when CF was either given 6 h before or simultaneously with CPA on day 2. However, when CF was given 6 h after CPA, a potentiated increase of embryolethality was observed at term and of structural chromosomal aberrations in embryos even before implantation, while sister chromatid exchange (SCE) frequency was less increased than after single CPA treatment. The results demonstrate the importance of pharmacokinetics in pregnancy even before implantation. Furthermore, since SCE may not only indicate DNA damage but also repair, the reduced SCE rate and the increase in structural chromosome aberrations suggest that CF can inhibit the repair of CPA-induced DNA damage in preimplantation embryos. Finally, for the first time the data are providing evidence that CF at subthreshold doses can potentiate the embryotoxicity of an alkylating agent in vivo even before implantation.

Studies on the Relation between Alkylating Agent‐Induced Chromosomal Aberrations and Embryotoxicity in Mice. II. The Potentiation of Teratogenicity and Clastogenicity of Isopropyl Methanesulfonate, N‐Methyl‐N‐Nitrosourea and Dimethyl Sulfate by Caffeine

ABSTRACT: The effects of caffeine on embryotoxicity including teratogenicity and clastogenicity of different classes of teratogens were examined in ICR‐mice in order to make clear the relationship between embryotoxicity and induced chromosomal aberrations in embryos. The teratogens used were isopropyl methanesulfonate (IPMS, 100 mg/kg), N‐methyl‐N‐nitrosourea (MNU, 20 mg/kg) and dimethyl sulfate (DMS, 25 mg/kg).In teratological studies, teratogens were given intraperitoneally on day 12 of gestation and caffeine subcutaneously in five divided doses (100 mg/kg for each) at 6 hours intervals during 0–24 hours following the teratogen treatment. In cyto‐genetical studies, teratogens (ip) were administered to ICR‐mice along with caffeine (sc) at a dose of 200 mg/kg on day 12 of gestation. Embryotoxicity was checked on day 18 of gestation, and clastogenicity at 6, 12, 18 and 24 hours following the co‐administration of these drugs.The co‐administration of caffeine and any one of these teratogens induced potentiative response in the formation of gross malformations and particularly in the production of chromosomal aberrations. The data obtained suggest the positive association of teratogenicity with clastogenicity of IPMS, MNU and DMS. Excess cell death due to chromosomal aberrations is highly suggestive as one of the mechanisms of embryotoxicity including teratogenicity of these teratogens.

Chromosome Aberrations in Embryos From In Vivo Aged Chicken Sperm

The effects of in vivo aging of spermatozoa on chromosome aberrations in early developing chick embryos were studied. There were aberrant chromosomes in all metaphase cells in one of 218 embryos examined or 0.458 percent for fresh sperm (1–3 days of fertile eggs) compared to seven of 315 embryos or 2.22 percent for aged sperm (9–14 days). Thus chromosome aberrations would account for about 25 percent of the expected embryonic death.

Chromosomal aberrations in embryos from overripe eggs

Overripeness in eggs of Rana pipiens was obtained by keeping postovulatory females at room temperature and delaying fertilization 3–5 days. In Xenopus laevis similar results were attained by delaying ovulation. About 80 crippled embryos, issued from overripe eggs that were used for chromosomal studies, had reached prefeeding stages ranging from 20 to 24. By this time the most severely abnormal individuals had already died, but also the majority of those studied would probably not have been able to survive until metamorphosis. Some malformed embryos still have normal chromosome complements, but the majority show deviations which are classified as meiotic, ploidic, and cleavage types. It is concluded that overripeness of the egg is an important cause of chromosomal nondisjunction in meiotic and in mitotic divisions. No definite relationship between the type of chromosomal deviation and the morphologic character of malformations has been established, so far.