Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification.

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.

Neonatal follow-up of 995 consecutively born children after embryo biopsy for PGD.

BACKGROUND: Outcome data on children born after assisted reproduction treatments are important for both patients and health-care providers. The objective of this study was to determine whether embryo biopsy as performed in PGD has an impact on the health of infants up to 2 months of age. METHODS: A prospective comparative follow-up study of children born after PGD and children born after ICSI by collecting written reports and performing a physical examination at 2 months was performed. Auxological data at birth and physical findings up to 2 months of age were compared for 995 children consecutively live born after embryo biopsy (1994-2009) and for a control group of 1507 children born after ICSI with embryo transfer on Day 5. RESULTS: No differences regarding mean term, prematurity (term <32 w and <37 w), mean birthweight, very low birthweight (<1500 g), perinatal death, major malformations and neonatal hospitalizations in singletons and multiples born following PGD versus ICSI were observed. Compared with ICSI, fewer multiples born following PGD presented a low birthweight (<2500 g) (P = 0.005). CONCLUSIONS: Embryo biopsy for PGD does not introduce extra risk to the overall medical condition of newborn children. Multiples born following embryo biopsy appear to be at lower risk for low birthweight compared with multiples born following ICSI.

PGD for a complex chromosomal rearrangement by array comparative genomic hybridization.

Patients carrying a chromosomal rearrangement (CR) have an increased risk for chromosomally unbalanced conceptions. Preimplantation genetic diagnosis (PGD) may avoid the transfer of embryos carrying unbalanced rearrangements, therefore increasing the chance of pregnancy. Only 7-12 loci can be screened by fluorescence in situ hybridization whereas microarray technology can detect genome-wide imbalances at the single cell level. We performed PGD for a CR carrier with karyotype 46,XY,ins(3;2)(p23;q23q14.2),t(6;14)(p12.2;q13) using array comparative genomic hybridization. Selection of embryos for transfer was only based on copy number status of the chromosomes involved in both rearrangements. In two ICSI-PGD cycles, nine and seven embryos were analysed by array, leaving three and one embryo(s) suitable for transfer, respectively. The sensitivity and specificity of single cell arrays was 100 and 88.8%, respectively. In both cycles a single embryo was transferred, resulting in pregnancy following the second cycle. The embryo giving rise to the pregnancy was normal/balanced for the insertion and translocation but carried a trisomy 8 and nullisomy 9 in one of the two biopsied blastomeres. After 7 weeks of pregnancy the couple miscarried. Genetic analysis following hystero-embryoscopy showed a diploid (90%)/tetraploid (10%) mosaic chorion, while the gestational sac was empty. No chromosome 8 aneuploidy was detected in the chorion, while 8% of the cells carried a monosomy for chromosome 9. In summary, we demonstrate the feasibility and determine the accuracy of single cell array technology to test against transmission of the unbalanced meiotic products that can derive from CRs. Our findings also demonstrate that the genomic constitution of extra-embryonic tissue cannot necessarily be predicted from the copy number status of a single blastomere.

Report on a consecutive series of 581 children born after blastomere biopsy for preimplantation genetic diagnosis.

BACKGROUND: Preimplantation genetic diagnosis (PGD) and subsequently preimplantation genetic screening (PGS) have been introduced since 1990. The difference from the already existing in vitro fertilization (IVF) technology, using intracytoplasmic sperm injection (ICSI), was the embryo biopsy at day 3 after fertilization. Although healthy children post-PGD/PGS have been born, the question of whether embryo biopsy could have any harmful effects has to be studied on large series in a prospective manner. METHODS: A prospective cohort study was undertaken from 1992 until 2005, using the same approach as for the follow-up of IVF and ICSI children conceived in the same centre. Questionnaires were sent to physicians and parents at conception and at delivery. Children were examined at 2 months of age by trained clinical geneticists whenever possible. RESULTS: Data collected on 581 post-PGD/PGS children showed that term, birthweight and major malformation rates were not statistically different from that of 2889 ICSI children, with overall rates of major malformation among these post-PGD/PGS and ICSI children being 2.13 and 3.38%, respectively (odds ratio [OR]: 0.62; exact 95% confidence limits [95% CL]: 0.31-1.15). However, the overall perinatal death rate was significantly higher among post-PGD/PGS children compared with ICSI children (4.64 versus 1.87%; OR: 2.56; 95% CL: 1.54-4.18). When stratified for multiple births, perinatal death rates among PGD/PGS singleton and ICSI singleton children were similar (1.03 versus 1.30%; OR: 0.83; 95% CL: 0.28-2.44), but significantly more perinatal deaths were seen in post-PGD/PGS multiple pregnancies compared with ICSI multiple pregnancies (11.73 versus 2.54%; OR: 5.09; 95% CL: 2.80-9.90). The overall misdiagnosis rate was below 1%. CONCLUSIONS: Embryo biopsy does not add risk factors to the health of singleton children born after PGD or PGS. The perinatal death rate in multiple pregnancies is such that both caution and long-term follow-up are required.

Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfers.

BACKGROUND: Human embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated. METHODS: In order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%). CONCLUSIONS: The clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.

Cumulative reproductive outcome after preimplantation genetic diagnosis: a report on 1498 couples.

BACKGROUND: Couples undergoing preimplantation genetic diagnosis (PGD) have a different background and set of treatment characteristics to couples undergoing regular IVF or ICSI. The aim of this study was to analyse the cumulative reproductive outcome of a large cohort of couples undergoing PGD in relation to a number of explanatory variables potentially affecting the prognosis. METHODS: Prospective cohort study, Kaplan-Meier analysis was performed to estimate real (observed) and expected (calculated) cumulative delivery rates, and Cox proportional hazard regression analysis was used to assess the effect of age, number of cumulus oocyte complexes collected, fertility status, parity, genetic category and method of pituitary suppression. RESULTS: Between 1993 and 2005, 2753 unselected consecutive cycles of ICSI and PGD were carried out in 1498 couples. The cumulative observed delivery rate overall per couple with a maximum of six treatment cycles of ICSI and PGD performed was 29%. The expected cumulative delivery rate (max six cycles) overall was 62%. There were no significant differences in cumulative delivery rates between the different genetic categories (i.e. availability of transferable embryos after PGD of 50 or 75%, chromosomal translocations or aneuploidy screening). The cumulative reproductive outcome in this PGD cohort was also not significantly affected by the fertility status of the couple, their parity or the method of pituitary suppression. However, the age of the patient and the number of oocytes contributed significantly to the reproductive results. CONCLUSION: This prospective observational study demonstrates that age has a significantly negative effect on outcome of PGD, due to poor reproductive performance of female partners 40 years of age and older. The number of oocytes collected has a significant and independent effect. The other factors studied did not affect the cumulative reproductive outcome in this PGD cohort.

Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer.

BACKGROUND: Single-embryo transfer is a well-accepted strategy to avoid multiple pregnancies in an assisted reproductive technology (ART) programme. Besides the morphological quality and embryo kinetics up to the blastocyst stage, preimplantation genetic screening (PGS) of aneuploidy has been advocated as an adjuvant approach to select the embryo. METHODS: Couples with a female partner younger than 36 were randomly assigned to undergo transfer of a single blastocyst in a cycle with or without PGS using FISH for the chromosomes X, Y, 13, 16, 18, 21, 22. RESULTS: After the enrolment of 120 of the projected 447 patients in each group, study recruitment was terminated prematurely on the basis of futility. The observed live birth delivery rates after ART were 30.8 versus 30.8% per randomized patient, 34.6 versus 34.6% per cycle initiated, 37.8 versus 37.0% per aspirated cycle and 41.6 versus 43.5% per embryo transfer for the control versus the PGS group, respectively, with absolute between-group differences (95% CI; P value) of 0% (-11.7 to 11.7; P = 1.00), 0% (-12.7 to 12.7; P = 1.00), -0.8% (-14.2 to 12.7; P = 0.91) and 2.1% (-12.7 to 16.7; P = 0.79), respectively. Even in this younger age group, only 61% of the embryos had a normal diploid status. CONCLUSIONS: The absence of a beneficial treatment effect in this randomized clinical trial provides no arguments in favour of PGS to improve live birth delivery rate following single-embryo transfer in women under the age 36. Clinical Trials.gov: NCT00670059.

Chromosomal aneuploidy in embryos conceived with unstimulated cycle IVF.

There is an ever increasing trend in reproductive medicine to reduce the intensity of ovarian stimulation for in vitro fertilization (IVF) and to restrict the number of embryos that are transferred into the uterine cavity. Recent findings suggest that the magnitude of ovarian stimulation affects the proportion of euploid embryos. As a result of the restriction in the number of embryos transferred, it becomes even more important to select the embryo with optimum implantational and developmental potential. Our aim was to asses the prevalence of numerical chromosomal abnormalities (aneuploidy) in unstimulated cycle IVF embryos. Thirty patients (mean age 31.4 years) underwent oocyte retrieval in a natural cycle without any form of ovarian stimulation, followed by intracytoplasmic sperm injection and Preimplantation genetic aneuploidy screening (PGS) for chromosomes X, Y, 13, 16, 18, 21 and 22. Out of 30 cycles, 21 oocytes were retrieved, 15 of which fertilized successfully. Eleven embryos developed sufficiently in order to undergo the PGS analysis, and four embryos proved to be aneuploid (36.4%; 95% CI: 10.9-69.2%). Six normal embryos were transferred in utero, resulting in three ongoing pregnancies. Two healthy girls were born and one patient miscarried. Numerical chromosomal abnormalities (aneuploidy) are present even in embryos of young women, and in the absence of ovarian stimulation.

Single embryo transfer in preimplantation genetic diagnosis cycles for women <36 years does not reduce delivery rate.

BACKGROUND: The Belgian legislation imposes single embryo transfer (SET) on women of <36 years in their first treatment cycle to avoid multiple pregnancies. The aim of this study is to assess the impact of this legislation on the outcome of preimplantation genetic diagnosis (PGD) for inherited diseases in young women undergoing SET. METHODS: A retrospective analysis of PGD cycles for monogenic disorders and translocations in women <36 years on their first treatment cycle. Two groups of patients were defined according to the implementation of the Belgian legislation: (i) double embryo transfer (DET), January 2001-June 2003 (ii) SET, July 2003-June 2005. The primary and secondary outcome measures were delivery per embryo transfer and multiple pregnancy rates, respectively. A subgroup analysis for monogenic disorders and translocations was performed. RESULTS: 62 cycles were included in the DET group and 73 cycles in the SET group. The mean age, number of cumulus-oocyte complexes, number of fertilized oocytes, number of biopsied and cryopreserved embryos were comparable between both groups. There was no significant difference in the delivery rates between the DET and the SET groups (33.9% versus 27.4%, respectively). Multiple pregnancies were avoided when SET was performed. When monogenic disorders and chromosomal translocations were separately evaluated, no significant difference in the delivery rate after SET was observed. CONCLUSIONS: The implementation of a SET policy in young women undergoing PGD for monogenic disorders and translocations enables a significant reduction of multiple pregnancies without significantly affecting the delivery rate.

Current value of preimplantation genetic aneuploidy screening in IVF.

Preimplantation genetic aneuploidy screening (PGS) has been performed during the last decade as a way of enhancing embryo selection in patients with an increased incidence of embryonic numerical chromosome abnormalities (advanced maternal age, recurrent miscarriage and recurrent implantation failure). It has been proposed that the replacement of euploid embryos in these patients would result in a higher implantation and pregnancy rate and a reduced miscarriage rate. Additionally, the transfer of fewer embryos could reduce the chances for multiple pregnancies in all IVF patients. Although, to date, multiple studies have addressed this issue, contradictory results have been encountered. As a result, the effectiveness of aneuploidy screening remains to be established. Moreover, child outcome studies documenting the safety of this procedure are needed. The aim of this review is to summarize the available evidence concerning the use of PGS to determine the current value of the technique.

Does PGD for aneuploidy screening change the selection of embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men?

BACKGROUND: An increased incidence of aneuploid embryos has been recently described from azoospermic men. The aim of this study was to assess if embryo selection on day 5, based on morphological criteria, would be different from the selection based on PGD for aneuploidy screening (AS) in couples undergoing ICSI for male azoospermia. METHODS: Sixty-two cycles of testicular sperm extraction (TESE)-ICSI with PGD-AS were included in the analysis. Two embryologists, blinded to the PGD-AS results, retrospectively reviewed the available embryology data from day 5 embryos and selected one, two or three embryos to be transferred. These results were compared with the selected embryos based on PGD-AS. RESULTS: A total of 39 cycles from non-obstructive azoospermia (NOA) and 23 cycles from obstructive azoospermia (OA) were retrospectively analysed. If single embryo transfer (SET) had been performed, in 64.8% of the NOA cycles and 54.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 10.8 and 36.6% of the cycles, respectively, an aneuploid embryo would have been chosen. If double ET (DET) had been performed, in 72.9% of the NOA cycles and 86.5% of the OA cycles, no difference in embryo choice would have occurred compared to PGD-AS and in 2.7 and 4.5% of the cycles, respectively, an aneuploid embryo would have been chosen. If triple ET (TET) had been performed, the outcome would have been the same as for DET. DISCUSSION: Our results suggest that under the terms of an SET policy, the performance of PGD-AS in azoospermia would result in a higher chance of success, as the possibility of selecting a euploid embryo is enhanced.

Which patients with recurrent implantation failure after IVF benefit from PGD for aneuploidy screening?

Patients with recurrent IVF failure are defined as patients who are younger than 37 years and who had at least three consecutive unsuccessful IVF/intracytoplasmic sperm injection (ICSI) cycles with good quality embryos. These patients might be predisposed to chromosome errors in their embryos and therefore might benefit from preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). This technique is, however, expensive and some normal embryos might be lost due to the error rate. The aim of this retrospective study was to define those patients who would benefit most from it. One hundred and twenty-one first PGD-AS cycles for recurrent IVF failure were analysed. The aneuploidy rate, 'no embryo transfer' rate, live birth rate per embryo transfer and implantation rate were respectively 48.3, 22.3, 29.7 and 19.5%. A multivariate logistic regression analysis gave us a predictive model demonstrating that to have a 90% probability of having an embryo transfer after PGD-AS, the patient should have at least 10 mature oocytes, eight normally fertilized oocytes and six embryos for biopsy. This study suggests that most patients with recurrent IVF failure may benefit from PGD-AS. Future studies, however, should more strictly define this heterogeneous group of patients, so that comparison is easier.

Comparison of the aneuploidy frequency in embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men.

BACKGROUND: The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with focal spermatogenesis in the testis (not sufficient to spill over into the ejaculate) may benefit from the technique. Previous reports suggest a higher pregnancy rate after ICSI treatment in patients with obstructive azoospermia (OA) compared to their non-obstructive azoospermia (NOA) counterparts, which could be due to a higher aneuploidy frequency in the embryos of the latter group. We therefore conducted a prospective cohort study to compare the aneuploidy frequency of the screened embryos between the two groups. METHODS: From May 2001 until September 2003, we offered couples with an OA or NOA partner ICSI in combination with preimplantation genetic diagnosis for aneuploidy screening. RESULTS: No difference in age (30.6 and 33.5 years) or stimulation parameters was observed between the two groups; 53 and 60% of the embryos were abnormal in the NOA and OA groups respectively (P = not significant). CONCLUSIONS: The aneuploidy frequency in embryos obtained from NOA as well as OA men is similar and very high, despite the young age of their female partners.

Can biological or clinical parameters predict testicular sperm recovery in 47,XXY Klinefelter's syndrome patients?

BACKGROUND: Contradictory results are available regarding prediction of testicular sperm extraction in 47,XXY patients. This study, therefore, aimed at assessing the availability of testicular sperm and evaluates clinical parameters predicting successful sperm retrieval in azoospermic 47,XXY Klinefelter's syndrome patients. METHODS: Sperm recovery procedures were performed in 50 non-mosaic azoospermic Klinefelter patients. The facial hair pattern and the presence of gynaecomastia in men with successful and unsuccessful sperm recovery were compared using Fisher's exact test. The predictive value of clinical parameters such as age, testicular volume, FSH, FSH:LH ratio, testosterone and androgen sensitivity index (LH x testosterone) for successful testicular sperm retrieval was evaluated using the receiver operating characteristics (ROC) curve analysis. RESULTS: In 24 patients (48%) testicular sperm were recovered. Ninety-four per cent of the men in whom sperm was found had a normal facial hair pattern compared to 93% in whom no sperm was recovered (not significant, NS). Seventeen percent of the men with successful testicular sperm extraction had gynaecomastia compared to 31% of the men with failed testicular sperm extraction (NS). The mean testicular volume of the largest testis in patients with sperm found was 4.2 ml compared to 3.6 ml in patients with no sperm found (NS). The mean FSH and testosterone values in patients with sperm recovered were 31.2 IU/l and 3.1 ng/ml versus 40.4 IU/l (P = 0.04) and 3.2 ng/ml (NS) in patients without sperm recovered. All examined clinical and biological parameters failed to predict the outcome of the testicular sperm extraction using ROC curve analysis. CONCLUSION: As in the general population of men with non-obstructive azoospermia, there are currently no clinical parameters predicting successful sperm retrieval in the subpopulation of patients with non-mosaic Klinefelter syndrome.

PGD in 47,XXY Klinefelter's syndrome patients.

The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with focal spermatogenesis in the testis, may benefit from the ICSI technique in order to father a child. As ICSI use has become more common, centres have introduced infertility treatment for Klinefelter patients. To date, 34 healthy children have been born using ICSI without PGD, and the conception of one 47,XXY fetus has been reported. In view of the possible risk of an increased gonosome number in the spermatozoa of Klinefelter patients, a safer approach--offering these couples ICSI combined with PGD--has been used, and has resulted in the birth of three healthy children. Couples in which the male suffered from Klinefelter's syndrome were first treated in 1995; these patients were offered ICSI + PGD using FISH technology, notably to enumerate the X and Y chromosomes. ICSI + PGD was performed in 32 cycles of 20 couples with spermatozoa originating from a fresh ejaculate (n = 1), testicular biopsy (n = 21) or frozen-thawed testicular biopsy (n = 10). Normal fertilization occurred in 56.0 +/- 22.4% of the successfully injected oocytes. On day 3 of development, 119 embryos from 29 cycles were of sufficient quality to undergo biopsy and subsequent PGD; a positive result was obtained in 113 embryos. Embryos were available for transfer in 26 cycles, with a mean of 1.6 +/- 0.6 embryos per transfer. Eight pregnancies were obtained, and five resulted in a delivery. A total of 113 embryos from couples with Klinefelter's syndrome was compared with 578 embryos from control couples with X-linked disease where PGD was used to determine gender. A significant fall occurred in the rate of normal embryos for couples with Klinefelter's syndrome (54.0%) compared with controls (77.2%). Moreover, a significantly increased risk of abnormalities was observed for sex chromosomes and autosomes; for each autosome separately, this reached significance level for chromosomes 18 and 21 only. Hence, a cautious approach is warranted in advising couples with non-mosaic Klinefelter's syndrome. Moreover, the use of ICSI + PGD or prenatal diagnosis should be carefully considered.

ESHRE preimplantation genetic diagnosis (PGD) consortium: data collection II (May 2000).

In 1997, the ESHRE PGD Consortium was formed as part of the ESHRE Special Interest Group on Reproductive Genetics, in order to undertake a long-term study of the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD). In December 1999, the first PGD Consortium report was published discussing referrals of 323 couples, 392 PGD cycles and 82 pregnancies and 79 children born. In the second round of data collection, contributing centres were asked to send in data from their PGD activities before January 1997, as well as from 1st October 1998 until 1st May 2000, in order to have as complete as possible an overview of PGD practices in these centres. A further 563 referrals were sent in as well as 926 PGD cycles, and data on 89 pregnancies (including seven pregnancies ongoing from the previous group) and 83 children were collected. This has led to a considerable amount of cumulative data being acquired: over a period of 7 years (the oldest PGD cycle reported dates from 1994), referral data on 886 couples, cycle data on 1318 PGD cycles and data on 163 pregnancies and 162 babies were collected. In all, these data are encouraging: they show first, that the practice of PGD is becoming more and more established, and an increasing number of different applications is emerging; and second, that collecting these data is worthwhile, as they will be a valuable source of information for all those involved, e.g. in counselling patients and interacting with governmental bodies.

The Brussels' experience of more than 5 years of clinical preimplantation genetic diagnosis.

This paper describes the 5 years' experience of preimplantation genetic diagnosis (PGD) at the Brussels Free University. Our first PGD was carried out in February 1993. Up to October 1998, we carried out 183 PGD cycles on fresh cleavage embryos of 92 couples for 25 different conditions. Patients were treated for autosomal recessive (n = 39), autosomal dominant (n = 65) and X-linked recessive (n = 47) monogenic disorders as well as for autosomal structural aberrations (n = 10), sex chromosome numerical and structural aberrations (n = 21) and a combination of the two latter (n = 1). Specific diagnosis was carried out by polymerase chain reaction (n = 108). Fluorescence in-situ hybridization was used for sexing (n = 64) and structural aberrations (n = 11). We transferred 1.6 +/- 1.1 embryos per cycle, resulting in an implantation rate of 12.0% per replaced embryo. Ongoing pregnancies were achieved in 29 cycles, i.e. 23 singletons, five twins and one dichorionic triplet with an acardius acranius. The ongoing pregnancy rates per cycle, per transfer and per couple were 16.4, 19.9 and 31.5% respectively. While 28 ongoing pregnancies resulted in the births of 34 infants, one pregnancy was terminated after misdiagnosis. The results of 24 PGD were confirmed by prenatal diagnosis or after birth while no information was available in four pregnancies. Our series demonstrates that PGD is a feasible technique by which to avoid the birth of genetically affected children to couples at risk.

Nuclear status and cytogenetics of embryos derived from in vitro-matured oocytes.

OBJECTIVE: To analyze embryos after in vitro maturation by investigating their nuclear status and cytogenetic constitution. DESIGN: Prospective randomized laboratory study. SETTING: Reproductive medicine unit in an academic hospital. PATIENT(S): Patients with male and tubal factor infertility undergoing fertility treatment. INTERVENTION(S): Denuded immature oocytes (n = 75) were matured in vitro for 24-30 hours, and intracytoplasmic sperm injection was performed 30 hours after oocyte retrieval. Fluorescence in situ hybridization was performed on the produced embryos. MAIN OUTCOME MEASURE(S): Blastomere content of the total embryo. RESULT(S): The in vitro-matured oocytes showed a similar fertilization rate as the in vivo-matured oocytes, but with a higher incidence of noncleavage (21.0%). In addition, 26.7% of these embryos arrested at the first mitotic division. Thirty embryos were processed for fluorescence in situ hybridization; only 6.7% had all mononuclear blastomeres, 30.0% had at least one binuclear blastomere, 43.3% had at least one multinuclear blastomere, and 56.6% contained anuclear cells. The chromosomal constitution was analyzed in 14 embryos, and chromosomal anomalies were found in 11 (78.5%). CONCLUSION(S): Germinal vesicle oocytes retrieved from superovulated patients and cultured in vitro for a short time had the ability to resume meiosis and achieve fertilization. However, arrest of embryo development was common. These embryos showed a high incidence of multinuclear blastomeres and aneuploidy, suggesting abnormal cytokinesis or genetic abnormalities.

Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) can be offered as an alternative to prenatal diagnosis (PND) to couples at risk of having a child with a genetic disease. The affected embryos are detected before implantation by fluorescent in situ hybridisation (FISH) for sexing (X-linked diseases) and chromosomal disorders (numerical and structural) or by polymerase chain reaction (PCR) for monogenic disorders (including some X-linked diseases). The accuracy and reliability of the diagnosis is increased by analysing two blastomeres of the embryo. However, the removal of two blastomeres might have an effect on the implantation capacity of the embryo. We have evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD cycles where a transfer was done. The patients were divided into five groups: a first group which received only embryos from which one cell had been removed, a second group which received only embryos from which two cells had been removed, a third group which received a mixture of embryos from which one and two cells had been taken, a fourth group where two and three cells had been removed, and a fifth group where three cells had been removed. The overall ongoing pregnancy rate per transfer was 26.1%, the overall implantation rate per transfer was 15.2% and the overall birth rate was 14.2%. Although pregnancy rates between the groups cannot be compared because the second group (two cells removed) contains more rapidly developing and therefore 'better quality' embryos, an ongoing pregnancy rate of 29.1% and an implantation rate of 18.6% per transferred embryo in this group is acceptable, and we therefore advise analysing two cells from a > or =7-cell stage embryo in order to render the diagnosis more accurate and reliable.

ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: preliminary assessment of data from January 1997 to September 1998. ESHRE PGD Consortium Steering Committee.

The first clinical application of preimplantation genetic diagnosis (PGD) was reported almost a decade ago. Since then, the range of genetic defects that can be detected at single cell level has increased dramatically. At the 13th Annual Meeting of ESHRE in Edinburgh in 1997, a PGD Consortium was formed to undertake the first systematic and long-term study of the efficacy and clinical outcome of PGD. We report here the first data collection covering the period of January 1997 to September 1998. Referral data on 323 couples have been collected for a variety of monogenic and chromosomal disorders, providing information about which patients, at risk for which genetic diseases, are interested in PGD. Data were collected on 392 PGD cycles, resulting in 302 embryo transfers and 66 clinical pregnancies. Because of the importance of follow-up of the children born after PGD, participating centres were asked to contribute data on the pregnancies achieved and the children born after PGD since the start of their PGD programme. Data on 82 pregnancies and 110 fetal sacs were collected, and information was available on 79 children. Finally, biopsy, fluorescence in-situ hybridization and polymerase chain reaction protocols were collected, clearly showing that no consensus exists on technical aspects such as which culture medium to use, and emphasizing the role the PGD Consortium could play in setting up guidelines for good laboratory practice. In conclusion, it is clear that the effort of gathering data on PGD cycles is worthwhile and will be continued in the future, preferably using electronic data collection.