Permanence of de novo segmental aneuploidy in sequential embryo biopsies.

STUDY QUESTION: Is de novo segmental aneuploidy (SA) a biological event or an artifact that is erroneously interpreted as partial chromosome imbalance? SUMMARY ANSWER: The detection of de novo SA in sequential biopsies of preimplantation embryos supports the biological nature of SA. WHAT IS KNOWN ALREADY: Although some SAs are detected in oocytes and in blastocysts, the highest incidence is observed in cleavage-stage embryos. Based on these findings, we can postulate that the majority of cells affected by SAs are eliminated by apoptosis or that affected embryos mainly undergo developmental arrest. STUDY DESIGN, SIZE, DURATION: This retrospective study includes 342 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between January 2014 and December 2018. Chromosome analysis was performed on 331 oocytes, 886 cleavage-stage embryos and 570 blastocysts (n = 1787). From 268 expanded blastocysts, the blastocoelic fluid (BF) was also analyzed (resulting in 2025 samples in total). In cases of SAs involving loss or gain in excess of 15 Mb, embryos were not considered for transfer and sequential biopsies were performed at following stages. This resulted in 66 sets where the initial diagnosis of SAs (4 made in polar bodies, 25 in blastomeres and 37 in trophectoderm (TE) cells) was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 2082 samples (2025 + 27 whole embryos) were processed by whole genome amplification followed by array comparative genomic hybridization. MAIN RESULTS AND THE ROLE OF CHANCE: The incidence of SAs was 6.3% in oocytes, increased to 16.6% in cleavage-stage embryos (P < 0.001) and decreased to 11.2% in blastocysts (P < 0.025 versus oocytes; P < 0.01 versus cleavage-stage embryos). The highest incidence of SAs was found in BFs (26.1%, P < 0.001). The analysis of 66 sets of sequential biopsies revealed that the initial finding was confirmed in all following samples from 39 sets (59.1% full concordance). In 12 additional sets, SAs were detected in some samples while in others the interested chromosome had full aneuploidy (18.2%). In three more sets, there was a partial concordance with the initial diagnosis in some samples, but in all TE samples the interested chromosome was clearly euploid (4.5%). In the remaining 12 sets, the initial SA was not confirmed at any stage and the corresponding chromosomes were euploid (18.2% no concordance). The pattern of concordance was not affected by the number of SAs in the original biopsy (single, double or complex) or by the absence or presence of concomitant aneuploidies for full chromosomes. LIMITATIONS, REASONS FOR CAUTION: Chromosome analyses were performed on biopsies that might not be representative of the true constitution of the embryo itself due to the occurrence of mosaicism. WIDER IMPLICATIONS OF THE FINDINGS: The permanence of SAs throughout the following stages of embryo development in more than half of the analyzed sets suggests for this dataset a very early origin of this type of chromosome imbalance, either at meiosis or at the first mitotic divisions. Since SAs remained in full concordance with the initial diagnosis until the blastocyst stage, a corrective mechanism seems not to be in place. In the remaining cases, it is likely that, as for full chromosome aneuploidy, mosaicism derived from mitotic errors could have occurred. In following cell divisions, euploid cell lines could prevail preserving the embryo chances of implantation. Due to the scarcity of data available, the transfer of embryos with SAs should be strictly followed up to establish possible clinical consequences related to this condition. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained. There are no conflicts of interest.

The educational and professional status of clinical embryology and clinical embryologists in Europe.

STUDY QUESTION: What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? SUMMARY ANSWER: It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. WHAT IS KNOWN ALREADY: Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. STUDY DESIGN, SIZE, DURATION: A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. MAIN RESULTS AND THE ROLE OF CHANCE: In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. LIMITATIONS, REASONS FOR CAUTION: Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: No.

Objective way to support embryo transfer: a probabilistic decision.

STUDY QUESTION: Is it feasible to identify factors that significantly affect the clinical outcome of IVF-ICSI cycles and use them to reliably design a predictor of implantation? SUMMARY ANSWER: The Bayesian network (BN) identified top-history embryos, female age and the insemination technique as the most relevant factors for predicting the occurrence of pregnancy (AUC, area under curve, of 0.72). In addition, it could discriminate between no implantation and single or twin implantations in a prognostic model that can be used prospectively. WHAT IS KNOWN ALREADY: The key requirement for achieving a single live birth in an IVF-ICSI cycle is the capacity to estimate embryo viability in relation to maternal receptivity. Nevertheless, the lack of a strong predictor imposes several restrictions on this strategy. STUDY DESIGN, SIZE, DURATION: Medical histories, laboratory data and clinical outcomes of all fresh transfer cycles performed at the International Institute for Reproductive Medicine of Lugano, Switzerland, in the period 2006-2008 (n = 388 cycles), were retrospectively evaluated and analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were unselected for age, sperm parameters or other infertility criteria. Before being admitted to treatment, uterine anomalies were excluded by diagnostic hysteroscopy. To evaluate the factors possibly related to embryo viability and maternal receptivity, the class variable was categorized as pregnancy versus no pregnancy and the features included: female age, number of previous cycles, insemination technique, sperm of proven fertility, the number of transferred top-history embryos, the number of transferred top-quality embryos, the number of follicles >14 mm and the level of estradiol on the day of HCG administration. To assess the classifier, the indicators of performance were computed by cross-validation. Two statistical models were used: the decision tree and the BN. MAIN RESULTS AND THE ROLE OF CHOICE: The decision tree identified the number of transferred top-history embryos, female age and the insemination technique as the features discriminating between pregnancy and no pregnancy. The model achieved an accuracy of 81.5% that was significantly higher in comparison with the trivial classifier, but the increase was so modest that the model was clinically useless for predictions of pregnancy. The BN could more reliably predict the occurrence of pregnancy with an AUC of 0.72, and confirmed the importance of top-history embryos, female age and insemination technique in determining implantation. In addition, it could discriminate between no implantation, single implantation and twin implantation with the AUC of 0.72, 0.64 and 0.83, respectively. LIMITATIONS, REASONS FOR CAUTION: The relatively small sample of the study did not permit the inclusion of more features that could also have a role in determining the clinical outcome. The design of this study was retrospective to identify the relevant features; a prospective study is now needed to verify the validity of the model. WIDER IMPLICATIONS OF THE FINDINGS: The resulting predictive model can discriminate with reasonable reliability between pregnancy and no pregnancy, and can also predict the occurrence of a single pregnancy or multiple pregnancy. This could represent an effective support for deciding how many embryos and which embryos to transfer for each couple. Due to its flexibility, the number of variables in the predictor can easily be increased to include other features that may affect implantation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant, CTI Medtech Project Number: 9707.1 PFLS-L, Swiss Confederation. No competing interests are declared.

ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:35-48.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHRE's recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos.

Predicting aneuploidy in human oocytes: key factors which affect the meiotic process.

BACKGROUND: To estimate the incidence of aneuploidy in relation to patients' characteristics, the type of hormonal stimulation and their response to induction of multiple follicular growth, 4163 first polar bodies (PB1s) were analyzed. METHODS: Five hundred and forty four infertile couples underwent 706 assisted conception cycles (640 with poor prognosis indications and 66 controls) in which chromosomal analysis of PB1 for the chromosomes 13, 15, 16, 18, 21 and 22 was performed. Results were evaluated in a multivariate analysis. RESULTS: The proportion of normal oocytes was directly correlated (P < 0.01) with (i) the number of mature oocytes and (ii) the establishment of a clinical pregnancy; and inversely correlated (P < 0.01) with (i) female age, (ii) causes of female infertility (endometriosis, abortions, ovulatory factor), (iii) poor prognosis indications (female age, number of previous cycles, multiple poor prognosis indications), (iv) number of FSH units per oocyte and (v) number of FSH units per metaphase II oocyte. There was a weak significance of frequency (P < 0.05) between type of abnormality (originated by chromatid predivision, chromosome non-disjunction or combined mechanisms in the same oocyte) and groups of the studied variables, rather than to a specific abnormality or a specific chromosome. CONCLUSIONS: The type of infertility had a significant effect on errors derived from the first meiotic division, whose incidence was significantly higher in the presence of endometriosis or of an ovulatory factor, and in women that experienced repeated abortions. Each aneuploidy event was found to be dependent not on a specific variable, but on groups of variables. In addition, the tendency of chromosomal abnormalities to occur simultaneously implies that the deriving aneuploidies can be of any type.

Paternal contribution to aneuploidy in preimplantation embryos.

The association between sperm indices and the chromosomal status of preimplantation embryos was assessed in 230 couples with a female partner younger than 36 years undergoing 295 cycles of preimplantation diagnosis for aneuploidy: 105 cycles had normozoospermic samples, 134 cycles presented with oligoasthenoteratozoospermia (OAT), while the remaining cycles had spermatozoa retrieved from the seminal tract due to obstructive (29 cycles) or non-obstructive azoospermia (NOA, 27 cycles). One blastomere was biopsied from day-3 embryos and analysed for chromosomes XY, 13, 15, 16, 17, 18, 21, and 22. From the testing of 1549 embryos, the proportion of chromosomally abnormal embryos was significantly lower in normozoospermic patients (55%) than in OAT (62%, P < 0.025) and NOA patients (69%, P < 0.005). Complex abnormalities were the most frequent defect in NOA (68%), which also demonstrated the highest incidence of gonosomal aneuploidy (12%). From the re-analysis of all blastomeres in 493 non-transferred embryos, 95% of NOA embryos were chaotic mosaics. In conclusion, a severe male infertility condition could contribute to the generation of chromosomal abnormalities in the resulting embryos. This might occur especially in NOA patients in which the high incidence of chromosomal abnormalities is mainly due to mosaicism and gonosomal aneuploidy.

Role of aneuploidy on embryo implantation.

Approximately 30% of oocytes in the human species carry a chromosomal imbalance. This condition has severe clinical consequences as approximately one-third of spontaneous abortions are aneuploid. The most obvious link to the increase of aneuploidy in oocytes is maternal age. This has been directly confirmed by the analysis of polar bodies. Their analysis permits to give confirmation of the high predisposition of oocytes to meiotic errors. Also, the study of chromosomes on sperm has revealed a frequency of 6-7% aneuploidy in normal sperm samples, and is significantly increased in cases of severe oligoasthenoteratospermia or azoospermia due to testicular failure. During the preimplantation period there is a progressive loss of abnormal embryos at specific stages in early development, through growth arrest and degeneration of abnormal embryos. The frequency of chromosomal abnormalities is strictly related to the category of patients (advanced maternal age, repeated cycles, altered karyotype, repeated miscarriages, TESE). Based on these considerations, preimplantation genetic diagnosis for aneuploidy is proposed in reproductive medicine with the finality of improving the clinical outcome after IVF. Substantial evidence has been accumulated on the positive impact of the technique, reporting increased implantation rates and a concomitant decrease of spontaneous abortions and trisomic pregnancies.

Oocyte euploidy, pronuclear zygote morphology and embryo chromosomal complement.

BACKGROUND: Pronuclear morphology has been proposed as an indicator of embryo development and chromosomal complement. In this study, the morphology of pronuclear zygotes generated from euploid oocytes [diagnosed by first polar body (PB1) analysis] was evaluated and compared with the configurations observed in chromosomally normal embryos (diagnosed by blastomere analysis). MATERIALS AND METHODS: Group 1--238 patients underwent 273 assisted conception cycles in combination with the screening of aneuploidy on PB1 for the chromosomes 13, 15, 16, 18, 21 and 22. Only normal oocytes were inseminated. Group 2--218 patients underwent 318 assisted conception cycles with aneuploidy screening on day 3 embryos. In both groups, oocytes were checked for fertilization and pronuclear morphology at 16 h after insemination. RESULTS: Seventy-three percent of zygotes from Group 1 had the configurations with centralized and juxtaposed pronuclei, large-size aligned or scattered nucleoli and PB located in the longitudinal or perpendicular axis of pronuclei. In Group 2, these configurations corresponded to those with the highest proportion of chromosomally normal embryos. Accordingly, in both groups, these configurations had a higher implantation rate than all the others. CONCLUSIONS: These observations confirm that some patterns of pronuclear morphology are associated with a higher proportion of euploidy and implantation reaffirming the relevance of this scoring system for the prediction of zygote viability.

Cryopreservation of biopsied embryos at the blastocyst stage.

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.

ESHRE PGD Consortium data collection IV: May-December 2001.

The ESHRE PGD Consortium was formed in 1997 to survey the practice of preimplantation genetic diagnosis (PGD). Since then, three reports have been published giving an overview on PGD from an ever-increasing number of centres and reporting on an increasing number of PGD cycles and pregnancies and babies born after PGD. After these initial influential publications, important shortcomings were identified primarily on the method of data collection, i.e. with Excel spreadsheets, and in the timing of the collection (cycles were collected in a different time frame from pregnancies and babies, making the follow-up of cycles very difficult). This is why the Steering Committee has made a major investment in developing and implementing a new database in FileMaker Pro 6. It was also decided that cycles would be collected from one calendar year, as well as the pregnancies and babies ensuing from that particular calendar year. This gave us the opportunity to take a closer look at the data collected earlier, and to attempt to improve their quality. This is a report on the corrected data from the first three data collections (I-III) as well as the result of the last data collection (IV) that was completely carried out using the new database.

Sperm aneuploidy and recurrent pregnancy loss.

Experiments of double target in-situ hybridization were performed separately for chromosomes 1-17, 8-18 and sex chromosomes on sperm samples from 20 couples suffering from three or more recurrent first trimester abortions. For a subset of this study population, additional experiments of multicolour fluorescence in-situ hybridization for chromosomes 4, 7, 12, 13, 15, 18, 21, and 22, were performed on the bases of the available data from abortive tissue karyotyping. A markedly high rate of sperm disomy (14.5-15.5%) was scored in only two cases. For three other patients, the cumulative disomy rates for chromosomes 1, 17, 8, 18, X and Y also increased but at a lower level (7.8-9.5%). For the remaining 15 patients, the frequency of sperm aneuploidy was moderately increased or normal. Men with recurrent pregnancy loss (RPL) and poor semen quality had baseline sperm aneuploidy and diploidy rates higher than men with normal semen parameters (with or without RPL). Using probes for chromosomes 1, 17, 8, 18, X and Y, significantly elevated frequencies of sperm aneuploidy (not diploidy) were found in 10% of men with a history of RPL. Their rate of sperm aneuploidy was 30-34%. For the other men, changes in sperm aneuploidy were not thought to affect RPL. Poor semen quality per se impacted negatively on sperm aneuploidy and diploidy, thus making the interpretation of clinical data more difficult.

Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching.

Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder. In this paper we describe a strategy optimized for preimplantation genetic diagnosis (PGD) of haemoglobinopathies combined with HLA matching. This procedure involves a minisequencing-based genotyping of HLA regions A, B, C and DRB combined with mutation analysis of the gene regions involved by mutation. Analysis of at least eight polymorphic short tandem repeat (STR) markers scattered through the HLA complex has also been included to detect potential contamination and crossing-over occurrences between HLA genes. The above assay can also be used for preimplantation HLA matching as a primary indication. The strategy was clinically applied for HLA matching in 17 cycles (14 for beta-thalassaemia, one for Wiscott-Aldrich syndrome and two for leukaemia). A reliable HLA genotype was achieved in 255/266 (95.9%) of the blastomeres. In total, 22 (14.8%) embryos were obtained that were HLA-matched with the affected siblings, 14 (9.4%) of which were unaffected and transferred back to the patients. Four clinical pregnancies were obtained, three of which (one twin, two singletons) are ongoing and were confirmed as healthy and HLA-identical with the affected children. Minisequencing-based HLA typing combined with HLA STR haplotyping has been shown to be a reliable strategy for preimplantation HLA matching. The major advantage of this approach is that the validation of a single assay can be done once and then used for the majority of the patients, reducing notably time needed for preclinical set-up of each case.

Prognostic role of preimplantation genetic diagnosis for aneuploidy in assisted reproductive technology outcome.

BACKGROUND: Preimplantation genetic diagnosis (PGD) for aneuploidy is recommended to couples at risk of generating chromosomally abnormal embryos. The aim of this study was to demonstrate that PGD for aneuploidy has an important role in the prognosis of subsequent treatments. METHODS: A total of 389 couples underwent their first PGD for aneuploidy due to either female age >or=38 years (n = 266) or >or=3 previous unsuccessful cycles (n = 123). After the first PGD followed by an unsuccessful treatment cycle, 141 couples underwent 175 subsequent PGD cycles. These patients were divided into three groups depending on the number of euploid embryos available for transfer in their first PGD cycle: group A included patients where no euploid embryos were diagnosed; group B included patients who had only one euploid embryo; and group C included patients with at least two normal embryos resulting from chromosomal analysis. RESULTS: In subsequent cycles, group A patients underwent significantly fewer transfers (45%) compared with group B (69%, P < 0.05) and group C patients (85%, P < 0.001). The pregnancy rate per transfer was significantly decreased in group A (15%) compared with group B (36%; P < 0.02) and group C (30%; P < 0.03). Accordingly, the live birth rate per patient was significantly lower in group A compared with group C (8.5% versus 30%; P < 0.005). CONCLUSIONS: The outcome of the first PGD for aneuploidy may have a predictive role for subsequent attempts.

Clinical value of preimplantation genetic diagnosis.

The clinical application of preimplantation genetic diagnosis (PGD) has provided an alternative approach for the prevention of affected pregnancies in couples at high reproductive risk. The frequent contribution of genetic factors to infertility problems makes PGD of particular value for assisted reproductive practices. In addition, the selection of euploid embryos for transfer has a strong impact on IVF efficiency as aneuploidies are the main cause of spontaneous abortions and implantation failures. In this study, the clinical outcome in PGD cycles is presented. The list of monogenic disorders for which PGD is performed is rapidly extending and the safety of the procedure has lead to an increasing interest among couples at high reproductive risk. Following PGD for aneuploidy, a higher implantation rate and a lower incidence of spontaneous abortions are obtained in patient categories where aneuploidy is a prominent cause of reproductive failure. In view of these findings, PGD has become an integral part of assisted reproductive techniques for the prevention of affected pregnancies and improvement of IVF efficiency.

The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders.

We have applied a new method of genetic analysis, called 'minisequencing', to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, beta-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods.

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.

Transvaginal ovarian drilling: a new surgical treatment for improving the clinical outcome of assisted reproductive technologies in patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate the efficacy of transvaginal ovarian drilling (TVOD) in patients with polycystic ovary syndrome (PCOS) who were undergoing IVF treatment. DESIGN: Pilot study. SETTING: Reproductive medicine unit. PATIENT(S): Eleven patients with PCOS undergoing treatment with assisted reproductive technology (ART). INTERVENTION(S): Selection criterion for TVOD was repeated poor performance in > or =2 previous IVF cycles. MAIN OUTCOME MEASURE(S): Controlled ovarian hyperstimulation parameters, number of eggs collected, fertilization rate, embryo cleavage rate, implantation rate, pregnancy rate compared with the cycles before TVOD. RESULT(S): In the cycle after TVOD, a significantly higher dosage of FSH was used (33.5 +/- 12 IU vs. 52.2 +/- 15 IU) to collect a higher number of oocytes in the presence of similar E2 values at the day of hCG administration. This resulted in significantly higher fertilization and cleavage rates (27% vs. 66% and 54% vs. 72%, respectively). The pregnancy and the implantation rates after TVOD were similar to those for normovulatory patients undergoing IVF for tubal factor infertility during the study period. CONCLUSION(S): Our data suggest that the TVOD is effective in improving IVF results in difficult to treat patients with PCOS, and it is less invasive and less expensive when compared with laparoscopic ovarian diathermy.

The in vivo and in vitro efficiency and efficacy of PGD for aneuploidy.

Preimplantation genetic diagnosis for aneuploidy was implemented on 1782 morphologically normal embryos generated in vitro by patients with a poor prognosis of pregnancy. Only 592 of them (34%) were diagnosed as chromosomally normal. Embryo transfer was accomplished in 240 cycles resulting in 79 clinical pregnancies (33%) and an implantation rate of 22.6%. The in vitro efficiency of the procedure was established by analysing all the blastomeres obtained from 311 non transferrable embryos and resulted to be 97.1%. The in vivo efficiency of the technique was calculated with the data derived from the prenatal diagnoses by examination of the infants at birth and was 97.8%. In consideration of the reported inaccuracy rate, patients are still recommended to undergo prenatal diagnosis. The transfer of PGD selected embryos in women of advanced reproductive age reduces by half the risk of having a trisomic pregnancy.