Upregulation of Oxidative Phosphorylation Genes in Cumulus Cells of The Polycystic Ovary Syndrome Patients with or without Insulin Resistance.

OBJECTIVE: The relationship between oxidative stress (OS), insulin resistance (IR), and polycystic ovary syndrome (PCOS) is an important medical issue in human reproduction. Some of the oxidative phosphorylation (OXPHOS) genes have been previously studied in granulosa and muscle cells of PCOS patients. Cumulus cells (CCs) remain close to the oocyte even after ovulation. This research has been designed to compare the expression of OXPHOS genes in CCs of PCOS, with or without insulin resistance. MATERIALS AND METHODS: In this experimental study, patients were included in PCOS insulin-resistant, PCOS insulinsensitive (IS), and control (fertile women with male infertility history) groups. The expression of NCF2, TXNIP, UCP2, NDUFB6, ATP5H, COX7C, NDUFA3, SDHA, and SDHB was studied by real-time polymerase chain reaction (PCR), and normalization was performed considering HPRT1 and CYC1 as reference genes. One-way ANOVA and Tukey test were used for data analysis. RESULTS: The results showed that the expression of NCF2, TXNIP, UCP2, and ATP5H was significantly higher in the IR group than IS and control groups (P<0.01). NDUFB6 showed the highest expression in the IS group, which was significantly different from other groups (P<0.01). The other genes of interest, except COX7C, were observed with the most transcriptional levels in the IS group, although there was no significant difference for those genes. CONCLUSION: Altered expression of genes involved in mitochondrial function compared to the control group in CCs of both IR and IS categories of the PCOS patients suggests that alteration in OXPHOS genes can contribute to the pathophysiology of PCOS.

Aneuploidy rate and Stemness in Low-level Mosaic Human Embryonic Stem Cells in the Presence/Absence of Bortezomib, Paclitaxel and Lapatinib.

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel and lapatinib was nontoxic at 0.01, 0.01, and 0.2µM concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

HOX cluster and their cofactors showed an altered expression pattern in eutopic and ectopic endometriosis tissues.

Endometriosis is major gynecological disease that affects over 10% of women worldwide and 30%-50% of these women have pelvic pain, abnormal uterine bleeding and infertility. The cause of endometriosis is unknown and there is no definite cure mainly because of our limited knowledge about its pathophysiology at the cellular and molecular levels. Therefore, demystifying the molecular mechanisms that underlie endometriosis is essential to develop advanced therapies for this disease. In this regard, HOX genes are remarkable because of their critical role in endometrial development and receptivity during implantation, which is attributed to their ability to mediate some of the sex steroid functions during the reproductive period. Access to the expression profiles of these genes would provide the necessary information to uncover new genes for endometriosis and assist with disease diagnosis and treatment. In this study we demonstrate an altered expression pattern for the HOX clusters (A-D) and their cofactors in both eutopic and ectopic conditions compared to control tissue biopsies. Remarkably, most of the intensive changes occurred in eutopic samples from endometriosis patients compared to control tissue biopsies. Pathway analysis revealed the involvement of differentially expressed genes in cancer that correlate with an association between endometriosis and cancer. Our results suggest critical roles for the HOX cluster and their cofactors in endometriosis pathophysiology.

Frequency of Sperm Aneuploidy in Oligoasthenoteratozoospermic (OAT) Patients by Comprehensive Chromosome Screening: A Proof of Concept.

BACKGROUND: Embryonic aneuploidy usually results in implantation failure and miscarriage. Considering significantly high frequency of sperm aneuploidy reported in oligoasthenoteratozoospermia (OAT) using fluorescence in situ hybridization (FISH) in limited number of chromosomes and lack of comprehensive chromosome screening (CCS) in OAT, the aim of this study was applying CCS in OAT sperm and comparison of the results with FISH findings. METHODS: Five OAT patients with normal blood karyotypes and history of implantation failure were included. The successfully amplified samples, each containing two sperm, were analyzed by array comparative genomic hybridization (aCGH). FISH was utilized mainly depending on the aneuploidies found by aCGH to assess their frequencies in total sperm population. RESULTS: In aCGH for 30 sperm, aneuploidy was found in 66% of samples. Following the study of 4300 sperm by FISH, an average of 55.46% aneuploidy was observed. No pregnancy was resulted with normal partners. CONCLUSION: Using aCGH, some abnormalities were observed that are not typically considered in sperm FISH studies. Despite small sample size of the comprehensive study, like other similar studies, the frequency of aneuploidies was considerable and similar to FISH. Aneuploidies revealed by aCGH at single sperm resolution were different from sperm population detected by FISH. Considering high frequency of aneuploidy in OATs sperm, preimplantation genetic testing for aneuploidy (PGT-A) can be used for in transfer of chromosomally normal embryos.

Lapatinib Decreases the Preimplantation Aneuploidy Rate of in vitro Fertilized Mouse Embryos without Affecting Completion of Preimplantation Development.

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (p < 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (p = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (p = 0.331 and p = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (p < 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.

Chromosomal instability reducing effect of paclitaxel and lapatinib in mouse embryonic stem cells with chromosomal abnormality.

Chromosomal abnormalities, as a frequent phenomenon in cultured embryonic stem cells (ESCs), is a major obstacle in the ESC application in regenerative medicine. Recent studies showed that aneuploid embryonic stem cells of humans and mice are more vulnerable to anticancer drugs, compared with normal cells. The aim of the current study was to evaluate effects of three anticancer drugs, paclitaxel, lapatinib and bortezomib, on mouse embryonic stem cells (mESCs) as a suitable and available model. To assess in vitro cell toxicity, two mESC lines were treated with the aforementioned drugs. Using G-band karyotyping and micronucleus assay, the effect of anticancer drugs in terms of reduction of chromosomal instability in the mESCs was evaluated in control and treatment groups. Also, apoptosis rate of both groups was estimated by FITC-Annexin V/Propidium Iodide (PI) double staining. In addition, the effect of these three drugs in maintaining the pluripotency was assessed through alkaline phosphatase assay and quantification of the expression of three key pluripotency genes, Nanog, Pou5f1 and Sox-2 was performed using Real Time PCR. The rate of numerical abnormalities after treatment with paclitaxel and lapatinib was lower than the control group. The expression level of pluripotency genes exhibited no significant difference between control and treatment groups. Administration of paclitaxel and lapatinib to the mESCs culture at an appropriate dose and in a timely manner could decrease chromosome stability without affecting pluripotency.

Noninvasive sexing of human preimplantation embryos using RT-PCR in the spent culture media: A proof-of-concept study.

Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium. Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique. In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test's sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively. Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.

Taxol Improves Pre-Implantation Development Potential of Mouse Embryos.

AIM: The objective of the present study was to investigate the development of mouse embryos and the chromosomal status after the pre-implantation treatment with paclitaxel (Taxol) based on the reports that indicate Taxol improves the developmental potential of vitrified human and mouse oocytes. METHODS: Outbred female mice were superovulated and in vitro fertilization (IVF) was carried out using sperms from the same strain. Two-cell stage mouse embryos were cultured in the presence of Taxol for 24 h. After the determination of a non-toxic dose of Taxol, embryo development in control and Taxol-treated groups was compared during 3.5 days post-IVF. The aneuploidy rate of embryos was assessed by fluorescence in situ hybridization for chromosomes 2 and 11. RESULTS: Development to morula and blastocyst stages was considerably enhanced following the addition of Taxol 0.01 µM compared to a similar situation in controls (p < 0.0001). Moreover, the degeneration rate was decreased following treatment with this concentration of Taxol (p < 0.01). The rate of aneuploidy in embryos and individual blastomeres did not vary between groups (p = 0.518 and 0.810 respectively). CONCLUSION: Pre-implantation treatment with Taxol 0.01 µM had a positive effect on the development to morula/blastocyst stages and decreased the degeneration rate without affecting the aneuploidy rate of chromosomes 2 and 11.

Chromosomal aberrations in pregnancy and fetal loss: Insight on the effect of consanguinity, review of 1625 cases.

BACKGROUND: Pregnancy loss affects 10%-15% of pregnancies and is caused by several factors, maternal and fetal. Most common cause is chromosomal aneuploidy and has traditionally been detected by karyotyping product of conception and/or fetal tissue. In recent years, array comparative genomic hybridization (a-CGH) has been used because of its higher detection and lower failure rates. METHODS: DNA was extracted from 1625 products of abortion or fetal tissue. In 1,104 cases both quantitative fluorescent-polymerase chain reaction (QF-PCR) and a-CGH, and in 521 cases only a-CGH, was performed. RESULTS: The detection rate using QF-PCR and a-CGH is 20% compared to 12.7%, overall, and 15.7%, excluding failed samples, by karyotypes in our center. QF-PCR and a-CGH failed in 1.9% of cases, while the failure rate for karyotypes was 20.1%. The difference of detection and failure rates is significant (p-value < 0.001 and p-value < 0.001 respectively). Unexpectedly we also found a significant difference in frequency of imbalances in related versus unrelated couples. (χ2  = 11.4926, p-value < 0.001). CONCLUSION: It is highly likely that the pregnancy loss in consanguineous couples is caused by other genetic and immune mechanisms. It is plausible that, through the same mechanism by which single gene disorders have a higher prevalence of manifesting disease in consanguineous couples, they can cause lethal genetic disorders leading to pregnancy loss and intra-uterine fetal death (IUFD) in these couples. Our findings suggest that this is a matter for further study as it will greatly influence the approach to counseling and managing consanguineous couples with pregnancy loss.

Association between The Number of Retrieved Mature Oocytes and Insulin Resistance or Sensitivity in Infertile Women with Polycystic Ovary Syndrome.

BACKGROUND: The objective of this study was to describe the association between luteinizing hormone (LH)/ follicle-stimulating hormone (FSH) ratio and demographic variables and maturation stage of oocytes in insulinresistant and insulin-sensitive patients with polycystic ovary syndrome (PCOS) in comparison with control group. MATERIALS AND METHODS: In this case-control study, 60 patients with in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) indication were subdivided into 3 groups as follow: 20 subjects were assigned to control (fertile women with male infertility history) group, 20 subjects with PCOS were insulin resistant (IR) and 20 subjects with PCOS were insulin sensitive (IS). After puncture, retrieved oocytes were classified into metaphase II (MII) as mature and in metaphase I (MI) or germinal vesicle stage (GV) as immature. Regression analyses were used to explore the association between MII oocyte number and demographic and clinical variables. RESULTS: LH/FSH ratio was significantly higher in PCOS-IR women compared to controls but not significantly different from that of PCOS-IS group. PCOS-IR women had lower MII oocyte number compared with that of controls. According to multiple regression analysis, the number of previous assisted reproductive technology (ART) cycles was negatively associated with the number of MII oocytes. CONCLUSION: Insulin resistance can be associated with reductions in MII oocyte number in patients with PCOS.

Origins of Intraindividual Genetic Variation in Human Fetuses.

BACKGROUND: Intraindividual copy number variation (CNV) origin is largely unknown. They might be due to aging and/or common genome instability at the preimplantation stage while contribution of preimplantation in human intraindividual CNVs occurrence is unknown. To address this question, we investigated mosaicism and its origin in the fetuses of natural conception. METHODS: We studied normal fetuses following therapeutic abortion due to maternal indications. We analyzed the genome of 22 tissues of each fetus by array comparative genomic hybridization for intraindividual CNVs. Each tissue was studied in 2 microarray experiments; the reciprocal aberrations larger than 40 Kb, identified by comparing tissues of each fetus, were subsequently validated using quantitative polymerase chain reaction. RESULTS: Through intraindividual comparison, frequency of reciprocal events varied from 2 to 9. According to the distribution pattern of the frequent CNV in derivatives of different germ layers, we found that its origin is early development including preimplantation, whereas CNVs with low frequency have occurred in later stages. Shared CNVs in both fetuses were belonged to thymus and related to the functional role of genes located in these CNVs. CONCLUSIONS: The origin of some of fetal CNVs is preimplantation stage. Each organ might inherit CNVs with an unpredictable pattern due to the extensive cell mixing/migration in embryonic development.

Downregulation of Extracellular Matrix and Cell Adhesion Molecules in Cumulus Cells of Infertile Polycystic Ovary Syndrome Women with and without Insulin Resistance.

OBJECTIVE: The extracellular matrix (ECM) of the cumulus oocyte complex (COC) is composed of several molecules that have different roles during follicle development. This study aims to explore gene expression profiles for ECM and cell adhesion molecules in the cumulus cells of polycystic ovary syndrome (PCOS) patients based on their insulin sensitivity following controlled ovarian stimulation (COS). MATERIALS AND METHODS: In this prospective case-control study enrolled 23 women less than 36 years of age who participated in an intracytoplasmic sperm injection (ICSI) program. Patients were subdivided into 3 groups: control (n=8, fertile women with male infertility history), insulin resistant (IR) PCOS (n=7), and insulin sensitive (IS) PCOS (n=8). We compared 84 ECM component and adhesion molecule gene expressions by quantitative real-time polymerase chain reaction array (qPCR-array) among the groups. RESULTS: We noted that 21 of the 84 studied genes differentially expressed among the groups, from which 18 of these genes downregulated. Overall, comparison of PCOS cases with controls showed downregulation of extracellular matrix protein 1 (ECM1); catenin (cadherin-associated protein), alpha 1 (CTNNA1); integrin, alpha 5 (ITGA5); laminin, alpha 3 (LAMA3); laminin, beta 1 (LAMB1); fibronectin 1 (FN1); and integrin, alpha 7 (ITGA7). In the IS group, there was upregulation of ADAM metallopeptidase with thrombospondin type 1 motif, 8 (ADAMTS8) and neural cell adhesion molecule 1 (NCAM1) compared with the controls (P<0.05). CONCLUSION: Downregulation of ECM and cell adhesion molecules seem to be related to PCOS. Gene expression profile alterations in cumulus cells from both the IS and IR groups of PCOS patients seems to be involved in the composition and regulation of ECM during the ovulation process. This study highlights the association of ECM gene alteration as a viewpoint for additional understanding of the etiology of PCOS.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.

Familial Case of Pelizaeus-Merzbacher Disorder Detected by Oligoarray Comparative Genomic Hybridization: Genotype-to-Phenotype Diagnosis.

Introduction. Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive hypomyelinating leukodystrophy characterized by nystagmus, spastic quadriplegia, ataxia, and developmental delay. It is caused by mutation in the PLP1 gene. Case Description. We report a 9-year-old boy referred for oligoarray comparative genomic hybridization (OA-CGH) because of intellectual delay, seizures, microcephaly, nystagmus, and spastic paraplegia. Similar clinical findings were reported in his older brother and maternal uncle. Both parents had normal phenotypes. OA-CGH was performed and a 436 Kb duplication was detected and the diagnosis of PMD was made. The mother was carrier of this 436 Kb duplication. Conclusion. Clinical presentation has been accepted as being the mainstay of diagnosis for most conditions. However, recent developments in genetic diagnosis have shown that, in many congenital and sporadic disorders lacking specific phenotypic manifestations, a genotype-to-phenotype approach can be conclusive. In this case, a diagnosis was reached by universal genomic testing, namely, whole genomic array.

The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells.

BACKGROUND: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. MATERIALS AND METHODS: In this cohort study, we used fluorescence in situ hybridi- zation (FISH) to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD) on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. RESULTS: Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%), while 2(6.7%) were diploid, and 2(6.7%) were triploid. Of those with mosaicism, 23(88.5%) were determined to be diploid-aneuploid and 3(11.5%) were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05); however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. CONCLUSION: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

The Relationship between HLA-G Gene Polymorphisms and Repeated Implantation Failure in Infertile Couples Undergoing Assisted Reproductive Technique.

 HLA-G is a tolerogenic molecule that expresses in cytotrophoblast cells and plays an important role in immune response suppression in maternal decidua. Interactions between the extracellular domains of the HLA-G protein with cell receptors of the immune system are well-known.This study investigated the association between HLA-G  gene  polymorphism  with repeated implantation failure (RIF). We used PCR followed by the sequencing technique for exons 2, 3, and 4, as well as intron 2 of the HLA-G gene in 100 couples with histories of two or more failed assisted reproductive technique (ART) attempts. The data were compared with the results of our previous study.The results indicated that some alleles of the HLA-G gene such as: 0106, 010106, 01010106 and 0105N (null) alleles were significantly higher in the patient group compared to the control group (p<0.05). There were higher SNPs at the +482 T/C and +506 -/C positions in failed ART couples compared to controls (p=0.03; p=0.01, respectively).HLA-G gene polymorphisms do not clearly affect the risk for implantation failure in most couples who undergo ART. However allelic variations, particularly in exons 3 and 4, and intron 2 of the HLA-G gene can lead to ART failure in human embryos.

DNA repair signalling pathway genes are overexpressed in poor-quality pre-implantation human embryos with complex aneuploidy.

OBJECTIVE: Chromosomal abnormalities and poor quality are correlated with DNA damage in the pre-implantation stage in humans. This study aimed to explore the altered expression of DNA damage signalling pathways - including apoptosis, cell cycle and DNA repair pathways - in poor-quality pre-implantation human embryos with complex aneuploidy. STUDY DESIGN: Surplus Day 4 embryos from candidates undergoing pre-implantation genetic screening were pooled into two groups. Group 1 included good-quality embryos that had simple aneuploidy, a single chromosome [according to fluorescence in situ hybridization-based pre-implantation genetic diagnosis (PGD) on Day 3], a normal rate of cell division, and graded as A or B (excellent to good). Group 2 included embryos with more than one aneuploid chromosome on PGD on Day 3, an abnormal rate of cell division, and graded as C or D (fair to poor). Gene expression of DNA damage signalling pathways was analysed using a real-time polymerase chain reaction-based array, which included 84 genes after specific pre-amplification of cDNA by a primer mix, including all array genes. RESULTS: In Group 2, five of the 84 genes studied showed significant overexpression (p<0.05): MSH3, XRCC1, RAD50, LIG1 and CDK7. Alterations were in agreement with genetic relationships in pathway analyses on DAVID. CONCLUSIONS: The five overexpressed genes are involved in DNA repair. Therefore, in comparison with cell cycle control and apoptotic pathways, DNA repair pathways are more activated in poor-quality pre-implantation human embryos with complex aneuploidy. This suggests that the dominant response to DNA damage in such embryos is DNA repair rather than cell division or apoptosis.

Self-correction of chromosomal abnormalities in human preimplantation embryos and embryonic stem cells.

Aneuploidy is commonly seen in human preimplantation embryos, most particularly at the cleavage stage because of genome activation by third cell division. Aneuploid embryos have been used for the derivation of normal embryonic stem cell (ESC) lines and developmental modeling. This review addresses aneuploidies in human preimplantation embryos and human ESCs and the potential of self-correction of these aberrations. Diploid-aneuploid mosaicism is the most frequent abnormality observed; hence, embryos selected by preimplantation genetic diagnosis at the cleavage or blastocyst stage could be partly abnormal. Differentiation is known as the barrier for eliminating mosaic embryos by death and/or decreased division of abnormal cells. However, some mosaicisms, such as copy number variations could be compatible with live birth. Several reasons have been proposed for self-correction of aneuploidies during later stages of development, including primary misdiagnosis, allocation of the aneuploidy in the trophectoderm, cell growth advantage of diploid cells in mosaic embryos, lagging of aneuploid cell division, extrusion or duplication of an aneuploid chromosome, and the abundance of DNA repair gene products. Although more studies are needed to understand the mechanisms of self-correction as a rare phenomenon, most likely, it is related to overcoming mosaicism.