Tsvia Frumkin

Female Sex Bias in Human Embryonic Stem Cell Lines

The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism.

Human embryonic stem cells carrying an unbalanced translocation demonstrate impaired differentiation into trophoblasts: an in vitro model of human implantation failure

Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosomal translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in situ hybridization and karyotype analysis were performed to analyze the t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring β-hCG secretion, by β-hCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of β-hCG and concomitantly expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of β-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in t(11;22)-hESCs, associated with implantation failure in unbalanced t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure.

Elucidation of abnormal fertilization by single-cell analysis with fluorescence in situ hybridization and polymorphic marker analysis

OBJECTIVE To analyze the genetic composition of oocytes and embryos presenting abnormal fertilization. DESIGN Case report. SETTING In vitro fertilization unit of a university-affiliated hospital. PATIENT(S) A couple with unexplained infertility with abnormal fertilizations in nine failed IVF-intracytoplasmic sperm injection cycles characterized by the presence of embryos with only one pronucleus and nonextrusion of the second polar body. INTERVENTION(S) All first polar bodies and blastomeres were analyzed by fluorescence in situ hybridization for chromosomes 13, 18, 21, X, and Y. Because some of the one-pronucleus embryos were found to be diploid, they were subjected further to single-cell polymerase chain reaction analysis with use of a panel of highly polymorphic markers from chromosomes 6, 7, 17, 19, X, and Y. MAIN OUTCOME MEASURE(S) Fluorescence in situ hybridization analysis of polar bodies and blastomeres and polymorphic marker analysis of day 5 embryos. RESULT(S) Fluorescence in situ hybridization analysis of the first polar body demonstrated normal segregation of chromosomes in meiosis I. Fluorescence in situ hybridization analysis of two aspirated blastomeres demonstrated three diploid and two mosaic triploid embryos. Polymorphic marker analysis, however, demonstrated that all embryos, including the diploid ones, had two sets of maternal alleles. CONCLUSION(S) Single-cell genetic analysis routinely used for preimplantation genetic diagnosis may provide insight into the genetic composition of oocytes and embryos. These data may be used in cases of abnormal fertilization or impaired embryo development for evidence-based fertility counseling regarding prognosis and treatment options.

Complex chromosomal rearrangement—a lesson learned from PGS

PurposeThe aim of the study is to report a case of non-diagnosed complex chromosomal rearrangement (CCR) identified by preimplantation genetic screening (PGS) followed by preimplantation genetic diagnosis (PGD) which resulted in a pregnancy and delivery of healthy offspring.MethodsA 29-year-old woman and her spouse, both diagnosed previously with normal karyotypes, approached our IVF-PGD center following eight early spontaneous miscarriages. PGS using chromosomal microarray analysis (CMA) was performed on biopsied trophectoderm. Fluorescence in situ hybridization (FISH), as well as re-karyotype, were performed on metaphase derived from peripheral blood of the couple. Subsequently, in the following PGD cycle, a total of seven blastocysts underwent CMA.ResultsA gain or loss at three chromosomes (3, 7, 9) was identified in six out of seven embryos in the first PGS-CMA cycle. FISH analysis of parental peripheral blood samples demonstrated that the male is a carrier of a CCR involving those chromosomes; this was in spite of a former diagnosis of normal karyotypes for both parents. Re-karyotype verified the complex translocation of 46,XY,t (3;7;9)(q23;q22;q22). Subsequently, in the following cycle, a total of seven blastocysts underwent PGD-CMA for the identified complex translocation. Two embryos were diagnosed with balanced chromosomal constitution. A single balanced embryo was transferred and pregnancy was achieved, resulting in the birth of a healthy female baby.ConclusionsPGS employing CMA is an efficient method to detect unrevealed chromosomal abnormalities, including complicated cases of CCR. The combined application of array CGH and FISH technologies enables the identification of an increased number of CCR carriers for which PGD is particularly beneficial.

Improving preimplantation genetic diagnosis (PGD) reliability by selection of sperm donor with the most informative haplotype

BackgroundThe study is aimed to describe a novel strategy that increases the accuracy and reliability of PGD in patients using sperm donation by pre-selecting the donor whose haplotype does not overlap the carrier’s one.MethodsA panel of 4–9 informative polymorphic markers, flanking the mutation in carriers of autosomal dominant/X-linked disorders, was tested in DNA of sperm donors before PGD. Whenever the lengths of donors’ repeats overlapped those of the women, additional donors’ DNA samples were analyzed. The donor that demonstrated the minimal overlapping with the patient was selected for IVF.ResultsIn 8 out of 17 carriers the markers of the initially chosen donors overlapped the patients’ alleles and 2–8 additional sperm donors for each patient were haplotyped. The selection of additional sperm donors increased the number of informative markers and reduced misdiagnosis risk from 6.00% ± 7.48 to 0.48% ±0.68. The PGD results were confirmed and no misdiagnosis was detected.ConclusionsOur study demonstrates that pre-selecting a sperm donor whose haplotype has minimal overlapping with the female’s haplotype, is critical for reducing the misdiagnosis risk and ensuring a reliable PGD. This strategy may contribute to prevent the transmission of affected IVF-PGD embryos using a simple and economical procedure.Trial registrationAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. DNA testing of donors was approved by the institutional Helsinki committee (registration number 319-08TLV, 2008). The present study was approved by the institutional Helsinki committee (registration number 0385-13TLV, 2013).