Altered gene expression of H19 and IGF2 in placentas from ART pregnancies.

INTRODUCTION: The aim of this study is to determine whether the gene expression and associated DNA methylation regulation of H19 and IGF2 are altered in placentas conceived by assisted reproductive technologies (ART) compared to natural conceptions. METHODS: 113 pregnancies were recruited resulting in 119 placentas (83 singletons and 36 twins), where 56 were conceived via in vitro fertilization (IVF), 41 via intracytoplasmic sperm injection (ICSI), and 22 naturally. Regulation of imprinting of H19 and IGF2 was determined by the DNA methylation status at three CpG sites within the H19 imprinting control region 1 (ICR1) using bisulphite pyrosequencing. Expression of H19 and IGF2 in 45 of these placentas (17 IVF, 14 ICSI, and 14 NC) was measured by determining the relative mRNA transcript levels using RT-qPCR in placental villi. RESULTS: Placental weight and birth weight were not significantly different between groups. H19 expression was significantly increased in both IVF and ICSI placentas when compared to controls (1.8 and 1.9 fold higher, respectively). Conversely, IGF2 was significantly decreased in both ART groups (0.8 and 0.7 fold lower, respectively). Mean DNA methylation at ICR1 was found to be similar between all groups. No correlation was found between DNA methylation at ICR1 and expression of either gene. However, a significant inverse relationship was found between H19 and IGF2 expression. CONCLUSION: We provide evidence of altered H19 and IGF2 expression in ART placentas. The altered expression pattern may suggest a loss of imprinting on the paternal allele. Furthermore, these alterations may not be entirely associated with DNA methylation at ICR1. We show further indirect evidence of the H19-IGF2 inverse expression pattern.

DNA methylation at H19/IGF2 ICR1 in the placenta of pregnancies conceived by in vitro fertilization and intracytoplasmic sperm injection.

DNA methylation at H19 ICR1 was investigated in the placenta from pregnancies conceived by in vitro fertilization, intracytoplasmic sperm injection, and from natural conception. Our results showed that there were no significant differences in mean methylation between all pregnancy groups; therefore, assisted reproductive technology may not affect proper imprinting of H19 and IGF2.

Sperm aneuploidy and meiotic sex chromosome configurations in an infertile XYY male.

BACKGROUND: There is little information regarding the behaviour of the extra Y chromosome during meiosis I in men with 47,XYY karyotypes and the segregation of the sex chromosomes in sperm. We applied immunofluorescent and FISH techniques to study the relationship between the sex chromosome configuration in meiotic germ cells and the segregation pattern in sperm, both isolated from semen samples of a 47,XYY infertile man. METHODS: The sex chromosome configuration of pachytene germ cells was determined by immunostaining pachytene nuclei for synaptonemal complex protein 3 (SCP3) and SCP1. FISH was subsequently performed to identify the sex chromosomes and chromosome 18 in pachytene cells. Dual- and triple-color FISH was performed on sperm to analyse aneuploidy for chromosomes 13, 18, 21, X, and Y. RESULTS: 46,XY/47,XYY mosaic pachytene cells were observed (22.2% vs. 77.8%, respectively). The XYY trivalent, and X+YY configurations were most common. While the majority of sperm were of normal chromosomal constitution, an increase in sex and autosome disomy was observed. CONCLUSIONS: The level of germ cell moscaicism and their meiotic sex chromosome configurations may determine sperm aneuploidy rate and fertility status in 47,XYY men. Our approach of immunostaining meiotic cells in the ejaculate is a novel method for investigating spermatogenesis in infertile men.

Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy.

Defects in early meiotic events are thought to play a critical role in male infertility; however, little is known regarding the relationship between early meiotic events and the chromosomal constitution of human sperm. Thus, we analyzed testicular tissue from 26 men (9 fertile and 17 infertile men), using immunofluorescent techniques to examine meiotic chromosomes, and fluorescent in situ hybridization to assess sperm aneuploidy. Based on a relatively small sample size, we observed that 42% (5/12) of men with impaired spermatogenesis displayed reduced genome-wide recombination when compared to the fertile men. Analysis of individual chromosomes showed chromosome-specific defects in recombination: chromosome 13 and 18 bivalents with only a single crossover and chromosome 21 bivalents lacking a crossover were more frequent among the infertile men. We identified two infertile men who displayed a novel meiotic defect in which the sex chromosomes failed to recombine: one man had an absence of sperm in the testes, while the other displayed increased sex chromosome aneuploidy in the sperm, resulting in a 45,X abortus after intracytoplasmic sperm injection. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. Recombination between the sex chromosomes may be a useful indicator for identifying men at risk of producing chromosomally abnormal sperm. An understanding of the molecular mechanisms that contribute to sperm aneuploidy in infertile men could aid in risk assessment for couples undergoing assisted reproduction.

Molecular and cytogenetic investigation of Y chromosome deletions over three generations facilitated by intracytoplasmic sperm injection.

BACKGROUND: The azoospermic factor (AZF) region is critical for normal spermatogenesis since microdeletions and partial deletions have been associated with infertility. We investigate the diagnostic ability of karyotyping in detecting clinically relevant Y chromosome deletions. The clinical significance of heterochromatin deletions, microdeletions and partial AZFc deletions is also evaluated. METHODS: A patient with a Yq deletion, affected by severe oligoasthenoteratozoospermia, underwent intracytoplasmic sperm injection (ICSI) which resulted in the birth of a healthy baby boy. The patient, his father and his son underwent Y chromosome microdeletion and partial AZFc deletion screening. We also studied the aneuploidy rate in the sperm of the patient by fluorescent in situ hybridization. RESULTS: AZF microdeletions were absent in the family. However, microdeletion analysis confirmed that the Yq deletion was limited to the heterochromatin. We found a partial AZFc gr/gr deletion in all three family members. We observed an increased rate of sex chromosome aneuploidy in the infertile patient. CONCLUSIONS: Cytogenetic analysis was misleading in identifying the Yq breakpoint. Infertility observed in the patient was associated with the gr/gr partial deletion. However, because of the incomplete penetrance of gr/gr deletions, the consequence of the vertical transmission of the deletion through ICSI remains unknown.