Preimplantation genetic diagnosis for gender selection in the USA.

Preimplantation genetic diagnosis (PGD) for gender selection for non-medical reasons has been considered an unethical procedure by several authors and agencies in the Western society on the basis that it could disrupt the sex ratio, that it discriminates against women and that it leads to disposal of normal embryos of the non-desired gender. In this study, the analysis of a large series of PGD procedures for gender selection from a wide geographical area in the USA shows that, in general, there is no deviation in preference towards any specific gender except for a preference of males in some ethnic populations of Chinese, Indian and Middle Eastern origin that represent a small percentage of the US population. In cases where only normal embryos of the non-desired gender are available, 45.5% of the couples elect to cancel the transfer, while 54.5% of them are open to have embryos transferred of the non-desired gender, this fact being strongly linked to cultural and ethnic background of the parents. In addition this study adds some evidence to the proposition that, in couples with previous children of a given gender, there is no biological predisposition towards producing embryos of that same gender. Based on these facts, it seems that objections to gender selection formulated by ethics committees and scientific societies are not well founded.

Preimplantation genetic diagnosis for complex chromosome rearrangements.

Complex chromosome rearrangements (CCR) are structural rearrangements that involve more than two breakpoints. The most common ones involve three chromosomes and three breakpoints, but double translocations can also occur. Theoretically, the potential for chromosome imbalance in the gametes of CCR carriers is higher than for simple translocations, and thus they are at an even higher risk of recurrent miscarriage. Preimplantation genetic diagnosis (PGD) has been shown to significantly reduce the risk of repeated pregnancy loss in translocation carriers, and thus it is well indicated for CCR. Here we describe different approaches for PGD of CCRs. After PGD of five couples carriers of CCR, only 6.4% of embryos were found normal or balanced, but the transfer of these six embryos into four couples, resulted in two pregnancies, one with normal non-identical twins, and the other with a balanced karyotype. Thus the implantation rate was 50%, and the pregnancy per retrieval was 40%, with 0% spontaneous abortions and 0% unbalanced offspring. This compares favorably with a prior history of spontaneous abortions, unbalanced offspring, and low fertility.

Follow-up of chimerism in children with hematological diseases after allogeneic hematopoietic progenitor cell transplants.

Using in situ hybridization with an X and Y chromosome probe mixture, 106 bone marrow samples from 38 patients with malignant and non-malignant hematological diseases who received sex-mismatched allogeneic hematopoietic progenitor cell transplants (PCT) in a single institution within short-term intervals (1, 3, 6, 12, 24 and >24 months) have been sequentially studied. The patients received either HLA-identical (n = 31) or non-identical (n = 7) PCT. Twenty-six children showed donor chimerism, 10 children showed mixed chimerism (MC) and two children presented autologous reconstitution. Chimerism status with different parameters has been related (age, sex, donor, disease status before PCT, conditioning regimen, GVHD prophylaxis, relapse, GVHD and survival). Our results indicate that female patients (P = 0.011) and a less intensive conditioning regimen (P = 0.039) are significantly associated with the MC status. Mixed chimerism is not, per se, significantly associated with leukemia relapse but an increase of the MC is indicative of clinical relapse.

Cytogenetic analysis using simultaneous and sequential fluorescence in situ hybridization.

A sequential fluorescence in situ hybridization (FISH) technique is described. This method allows the detection of up to eighteen chromosome pairs in consecutive hybridizations (8 steps) on the same metaphase using centromeric, whole chromosome painting, and single copy DNA probes with different fluorochromes. The technique may be used with diagnostic purposes in cases with poor cytogenetic material.

Outcome of preimplantation genetic diagnosis of translocations.

OBJECTIVE: To review 35 cases of preimplantation genetic diagnosis (PGD) of translocations with several methods, including telomeric probes. DESIGN: Retrospective study. SETTING: Clinical IVF laboratory. PATIENT(S): Thirty-five couples with one partner carrying a chromosomal translocation. INTERVENTION(S): PGD of translocation after polar-body or embryo biopsy. MAIN OUTCOME MEASURE(S): Pregnancy outcome. RESULT(S): Several trends were observed. First, PGD can achieve a statistically significant reduction in spontaneous abortion, from 95% to 13%. Second, the chances of achieving pregnancy are correlated with 50% or more of the embryos being chromosomally normal. Third, patients with robertsonian translocations produced fewer abnormal gametes and more pregnancies than did patients with reciprocal translocations. Fourth, a new fluorescence in situ hybridization protocol for PGD of translocations, which involves applying telomeric probes, has proved adequately reliable with a 6% average error rate. CONCLUSION(S): PGD of translocations achieves a statistically significant reduction in spontaneous abortion, both for polar-body and blastomere biopsy cases. Pregnancy outcome depended on the number of normal embryos available for transfer, with patients having <50% abnormal embryos achieving the most pregnancies. Because robertsonian translocations caused fewer abnormal embryos than reciprocal translocations, they also resulted in higher rates of implantation.

Current awareness

In order to keep subscribers up-to-date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of prenatal diagnosis. Each bibliography is divided into 17 sections: 1 Books, Reviews & Symposia; 2 General Interest; 3 Normal Fetal Development; 4 Gametogenesis and Pre-implantation Diagnosis; 5 First Trimester Diagnosis; 6 Second Trimester Diagnosis; 7 Fetal Diagnosis by Ultrasound and Other Imaging; 8 Maternal Screening; 9 Screening for Carriers of Genetic Abnormality; 10 Technological Developments; 11 Confined Placental Mosaicism and Uniparental Disomy; 12 Molecular Cytogenetics; 13 Fetal Cells in Maternal Circulation; 14 Fetal Therapy; 15 Psychosocial Aspects; 16 Epidemiology and Environmental Factors; 17 Developmental Pathology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted

Wide range of chromosome abnormalities in the embryos of young egg donors.

Embryo chromosome studies show high rates of abnormalities, above 50%, but most embryos studied were from patients aged 35 and older. The objectives of this study were firstly, to evaluate the rate of chromosome abnormalities in embryos from young egg donors, and secondly, to compare the range of chromosome abnormality rates between donors and non-egg donor cycles, both undergoing preimplantation genetic diagnosis (PGD) for infertility using fluorescence in-situ hybridization analysis with probes for chromosomes X, Y, 13, 15, 16, 18, 21, and 22. On average, only 43% of the embryos were chromosomally normal, while the comparison group had euploidy rates between 34 (age group 18-34) (P < 0.001) and 21% (age group 40-45) (P < 0.001). There was considerable variation between donor cycles, with almost one-third having less than 30% normal embryos. Also, within donors and recipients repeating several IVF cycles with PGD, only 29-56% of the second PGD cycles had similar rates of normal embryos to the first cycle, while in the comparison group it was 64%. The results can explain why some egg donors are successful whereas others are not, and may also show that a policy of PGD for first time egg donors is appropriate and indicated.

Chromatid segregation analysis in native human lymphocyte anaphases using sequential fluorescence in situ hybridization.

A sequential multiprobe fluorescence in situ hybridization technique was developed to study the 13, 18, 21, X and Y chromatid segregation in human lymphocytes anaphases cultures without antimitotic treatment. This method was used to know if exist any different chromosomes segregation in lymphocytes from Down syndrome patients and compared it with controls. The results show that the prevalent sequence of centromere separation was X, 13, 21, Y and 18 in Down syndrome patients and Y, 13, X, 21 and 18 in controls. Chromatid segregation in early anaphase was asynchronic for all chromosome pairs studied. Late anaphase showed a frequency of non-disjunction of 4.5% in the controls, affecting only chromosomes 18 and Y; in the Down syndrome patients, the frequency was higher (20.3%) and affected all chromosomes studied. This technique could be applicated to know the incidence of non disjunction in couples with repetitive abortions or in cases with different aneuploidies in the offspring.

Female gamete segregation in two carriers of translocations involving 2q and 14q.

FISH, using a combination of whole-chromosome painting and telomeric probes, was used to study the gamete segregation of two female carriers of translocations involving the same chromosome arms, 2q and 14q. Preimplantation genetic diagnosis of the first polar bodies of these oocytes permitted selecting normal embryos for replacement.

Improvements of preimplantation diagnosis of aneuploidy by using microwave hybridization, cell recycling and monocolour labelling of probes.

Recent studies indicate that preimplantation genetic diagnosis of aneuploidy significantly reduces spontaneous abortions and increases pregnancy rates in women of advanced maternal age undergoing in-vitro fertilization. A new protocol was developed involving cell recycling (sequential hybridizations) and microwave hybridization of repetitive probes in order to obtain in a few hours enumeration results for chromosomes X, Y, 1, 13, 15, 16, 18, 21 and 22 in single blastomeres. The error rate using this protocol was significantly lower than in previous protocols and the analysis of nine chromosomes can be achieved in a shorter time than before.

Analysis of chromosome abnormalities in sperm and embryos from two 45,XY,t(13;14)(q10;q10) carriers.

Robertsonian translocation t(13q14q) is studied in sperm and embryos of two couples undergoing preimplantation genetic diagnosis (PGD) in which both males are carriers of the translocation. It is already known that the chances of achieving pregnancy for a translocation carrier are directly linked to the number of normal or balanced embryos available for replacement. In our work it was found that the frequency of balanced spermatozoa was almost identical in both patients (74 and 77%), and after PGD, the frequencies of abnormal embryos caused by the translocation were also similar. Sperm chromosome analysis in translocation carriers can provide a reasonable basis for estimating a baseline of chromosome abnormalities to be found in embryos during an assisted reproductive cycle. However, individual factors not linked to the translocation can also produce other chromosome abnormalities (mosaicism, haploidy, polyploidy) and may compromise the chances of achieving a viable pregnancy.

Gamete segregation in female carriers of Robertsonian translocations.

Eleven female carriers of either 45,XX,der(13;14) (q10;q10) or 45,XX, der(14;21)(q10;q10) underwent hormonal stimulation with the purpose of producing enough oocytes for in-vitro fertilization and preimplantation genetic diagnosis. Polar body biopsy was performed in those oocytes and FISH with painting probes was applied in their metaphase-like first polar body chromosomes. In this way, unbalanced, normal and balanced oocytes could be distinguished and segregation modes ascertained. der(14;21)(q10;q10) produced 42% unbalanced, 37% normal and 21% balanced oocytes (n = 86) while der(13;14)(q10;q10) generated 33% unbalanced, 51% normal and 16% balanced oocytes (n = 69). In both translocations the number of normal oocytes was significantly higher than the number of balanced oocytes. However, while the frequency of unbalanced events involving chromosome 13 and 14 was similar in der(13;14)(q10;q10), there were significantly more abnormalities involving chromosome 21 than 14 in the der(14;21) (q10;q10) cases. When comparing survival rates to term, trisomies from Robertsonian origin seem to survive more often than those originated by non-disjunction in non-translocation carriers. The meiotic segregation patterns found in female Robertsonian translocations are different from those described in male carriers, with higher rates of unbalanced gametes in females than in males.

Quantitative determinations of chemical compounds with nutritional value from Inca crops: Chenopodium quinoa ('quinoa').

Quantitative determinations of total and soluble proteins, total and free sugars, starch, total lipids, tanins, ash (Ca, Na, K, Fe, and P), and caloric value were carried out on quinoa flour. Results show that the amount of soluble proteins was higher than the standard value for wheat and maize and was very close to that of barley's. The yield of free sugars like glucose (4.55%), fructose (2.41%) and sucrose (2.39%) were also of importance. Iron and calcium levels were higher than the reported values for maize and barley. The same occurred for the caloric value (435.5 Kcal/100 g). The content of saponins was also examined since its effect on red blood cells of group A and O has been related as a potential problem of the Andes population. From the chemical analysis a more complete view about quinoa as human food was presented.

Preimplantation genetic diagnosis of pericentric inversions.

Inversions are structural chromosome abnormalities that may be associated with infertility, multiple miscarriage and chromosomally unbalanced offspring. Preimplantation genetic diagnosis (PGD) with subtelomeric probes was used to select for transfer only those embryos that were normal or balanced for three pericentric inversions. In contrast to previous protocols the present procedure allows the detection of unbalanced embryos that might arise from U-recombination in the inverted region. Additionally, aneuploidy screening was carried out in two cases by a second round of fluorescent in situ hybridization (FISH) with centromeric probes. Of the three couples that underwent the procedure one became pregnant twice. The first pregnancy delivered a healthy and chromosomally normal baby and the second pregnancy is ongoing with triplets.

A case with 47,XXY,del(15)(q11;q13) karyotype associated with Prader-Willi phenotype.

Herein we present the case of a 12-year-old boy who attended our clinic for obesity and hyperphagia. As a newborn he was noted to have diffuse muscular hypotonia and poor sucking response. At the age of 11 years, he was admitted to hospital for respiratory insufficiency. He had personality disorders characterized by temper tantrums and violent outbursts including self-mutilation. Physical evaluation revealed marked central obesity, he had small hands and feet, and also genital hypoplasia. Of the biochemical parameters, hyperglycemia and a low serum testosterone level must be emphasized. The patient fulfills the clinical criteria of typical Prader-Willi syndrome. Cytogenetic and fluorescence in situ hybridization analysis showed a karyotype 47,XXY, del(15)(q11;q13). To our knowledge this is the first report of the aforementioned genotype expressed as Prader-Willi phenotype in childhood.

Possible interchromosomal effect in embryos generated by gametes from translocation carriers.

BACKGROUND: The incidence of abnormal pregnancies in carriers of balanced translocations depends strictly on the chromosomes involved in the translocations. The aim of this study was to verify whether conventional aneuploidy screening could be advantageously combined with preimplantation genetic diagnosis (PGD) for translocations. METHODS: Twenty-eight carriers of Robertsonian and reciprocal translocations underwent 43 PGD cycles; specific probes were used to screen the translocation in 172 embryos generated by 35 cycles; most of these embryos were also screened for chromosomes 13, 16, 18, 21, 22 (n = 166), XY (n = 107), 1 (n = 17) and 15 (n = 88). For the remaining eight cycles (carriers of reciprocal translocations) only the chromosomes involved in common aneuploidy screening were investigated on the 40 embryos generated in vitro. RESULTS: In Robertsonian translocations, the proportion of embryos with abnormalities due to the translocation was 21%, common aneuploidies contributed 31% of total abnormalities, whereas the remaining 36% of embryos had abnormalities due to both types of chromosome. For reciprocal translocations, the chromosomes involved in the translocation were responsible for 65% of total abnormalities; only 6% of the embryos were abnormal for common aneuploidies and 16% carried abnormalities due to both the chromosomes involved in the translocation and those not related to the translocation. CONCLUSIONS: An interchromosomal effect seems to play a role in the case of Robertsonian translocations, where the relevant contribution of aneuploidy exposes the couple to an additional risk of abnormal pregnancy.

Double locus analysis of chromosome 21 for preimplantation genetic diagnosis of aneuploidy.

Preimplantation genetic diagnosis (PGD) of numerical chromosome abnormalities significantly reduces spontaneous abortions and may increase pregnancy rates in women of advanced maternal age undergoing in vitro fertilization. However, the technique has an error rate of around 10% and trisomy 21 conceptions have occurred after PGD. To further reduce the risk of transferring trisomy 21 embryos to the patient, we designed a protocol that analyzes chromosome 21 twice by targeting two different loci. This protocol was applied to 388 embryos from 60 cycles of PGD of aneuploidy. The scoring criterion used was based on giving equal importance to both probe results. Of the 242 embryos diagnosed as abnormal, 125 were re-biopsied to assess the rate of false positives and false negatives of the protocol and their clinical relevance. The results of the present study showed no reduction in the overall fluorescent in situ hybridization (FISH) error rate for single cells. However, by using a different scoring criterion, the incidence of false negative can be reduced to 1.6% without missing any trisomy 21. In addition, the present study suggests that if two or more loci from the same chromosome could be simultaneously analyzed in single cells, errors caused by false monosomies could be reduced.

Predictability of preimplantation genetic diagnosis of aneuploidy and translocations on prospective attempts.

The aim of this study was to determine if the outcomes of aneuploidy and translocation testing by preimplantation genetic diagnosis (PGD) at the 8-cell stage have a predictive value for new genetic diagnosis cycles. In total, 83 cycles (39 patients) undergoing PGD of translocations and 378 cycles (176 patients) of aneuploidy were included. Predictability, defined as having similar rate (+/-20%) of euploid embryos in the first and successive cycles, was found in 66% of patients undergoing aneuploidy testing. Predictability was found significantly more often in patients undergoing PGD of translocations (90%, P = 0.006). In addition, patients with 0, <30 or > or =30% euploid embryos in the first cycle were compared and groups 0 and <30% had significantly fewer euploid embryos in the second cycle (22-26%) than those of the group with > or =30% (37%) (P < 0.05). Patients who did not become pregnant after the first attempt were stimulated more aggressively than those becoming pregnant, producing significantly more embryos in the second than in the first cycle (P < 0.001). Therefore, correlation between euploidy rate and pregnancy rate could not be assessed objectively between cycles. In conclusion, the PGD results of a first cycle can predict the results of the second cycle, but this is likely to be of more value when the condition investigated is translocation rather than aneuploidy. The chance of pregnancy is usually related to the number of euploid embryos.