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Study Question: Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes?, Summary Answer: The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes., What Is Known Already: The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood., Study Design, Size, Duration: As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 μl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified., Participants/materials, Setting, Methods: In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy., Main Results and the Role of Chance: The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy., Limitations, Reasons for Caution: The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases., Wider Implications of the Findings: This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies., Study Funding/competing Interest(s): This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study., Trial Registration Number: ClinicalTrials.gov (NCT03520933)., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: How well can whole chromosome copy number analysis from a single trophectoderm (TE) biopsy predict true mosaicism configurations in human blastocysts?, Summary Answer: When a single TE biopsy is tested, wide mosaicism thresholds (i.e. 20-80% of aneuploid cells) increase false positive calls compared to more stringent ones (i.e. 30-70% of aneuploid cells) without improving true detection rate, while binary classification (aneuploid/euploid) provides the highest diagnostic accuracy., What Is Known Already: Next-generation sequencing-based technologies for preimplantation genetic testing for aneuploidies (PGT-A) allow the identification of intermediate chromosome copy number alterations potentially associated with chromosomal mosaicism in TE biopsies. Most validation studies are based on models mimicking mosaicism, e.g. mixtures of cell lines, and cannot be applied to the clinical interpretation of TE biopsy specimens., Study Design, Size, Duration: The accuracy of different mosaicism diagnostic thresholds was assessed by comparing chromosome copy numbers in multiple samples from each blastocyst. Enrolled embryos were donated for research between June 2019 and September 2020. The Institutional Review Board at the Near East University approved the study (project: YDU/2019/70-849). Embryos showing euploid/aneuploid mosaicism (n = 53), uniform chromosomal alterations (single or multiple) (n = 25), or uniform euploidy (n = 39) in their clinical TE biopsy were disaggregated into five portions: the inner cell mass (ICM) and four TE segments. Collectively, 585 samples from 117 embryos were analysed., Participants/materials, Setting, Methods: Donated blastocysts were warmed, allowed to re-expand, and disaggregated in TE portions and ICM. PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion Reporter software to estimate raw chromosome copy numbers. Intra-blastocyst comparison of copy number data was performed employing different thresholds commonly used for mosaicism detection. From copy number data, different case scenarios were created using more stringent (30-70%) or less stringent criteria (20-80%). Categorical variables were compared using the two-sample z test for proportions., Main Results and the Role of Chance: When all the five biopsies from the same embryo were analysed with 30-70% thresholds, only 8.4% (n = 14/166) of patterns abnormal in the original analysis revealed a true mosaic configuration, displaying evidence of reciprocal events (3.6%, n = 6/166) or confirmation in additional biopsies (4.8%, n = 8/166), while most mosaic results (87.3% of total predicted mosaic patterns) remained confined to a single TE specimen. Conversely, uniform whole chromosome aneuploidies (28.3% of total patterns, n = 47/166) were confirmed in all subsequent biopsies in 97.9% of cases (n = 46/47). When 20-80% thresholds were employed (instead of 30-70%), the overall mosaicism rate per biopsy increased from 20.2% (n = 114/565) to 40.2% (n = 227/565). However, the use of a wider threshold range did not contribute to the detection of additional true mosaic patterns, while significantly increasing false positive mosaic patterns from 57.8% to 79.5% (n = 96/166; 95% CI = 49.9-65.4 vs n = 271/341; 95% CI = 74.8-83.6, respectively) (P < 0.00001). Moreover, the shift of the aneuploid cut-off from 70% to 80% of aneuploid cells resulted in mosaicism overcalling in the high range (50-80% of aneuploid cells), impacting the accuracy of uniform aneuploid classification. Parametric analysis of thresholds, based on multifocal analysis, revealed that a binary classification scheme with a single cut-off at a 50% level provided the highest sensitivity and specificity rates. Further analysis on technical noise distribution at the chromosome level revealed a greater impact on smaller chromosomes., Limitations, Reasons for Caution: While enrolment of a population enriched in embryos showing intermediate chromosome copy numbers enhanced the evaluation of the mosaicism category compared with random sampling such study population selection is likely to lead to an overall underestimation of PGT-A accuracy compared to a general assessment of unselected clinical samples. This approach involved the analysis of aneuploidy chromosome copy number thresholds at the embryo level; future studies will need to evaluate these criteria in relation to clinical predictive values following embryo transfers for different PGT-A assays. Moreover, the study lacked genotyping-based confirmation analysis. Finally, aneuploid embryos with known meiotic partial deletion/duplication were not included., Wider Implications of the Findings: Current technologies can detect low-intermediate chromosome copy numbers in preimplantation embryos but their identification is poorly correlated with consistent propagation of the anomaly throughout the embryo or with negative clinical consequences when transferred. Therefore, when a single TE biopsy is analysed, diagnosis of chromosomal mosaicism should be evaluated carefully. Indeed, the use of wider mosaicism thresholds (i.e. 20-80%) should be avoided as it reduces the overall PGT-A diagnostic accuracy by increasing the risk of false positive mosaic classification and false negative aneuploid classification. From a clinical perspective, this approach has negative consequences for patients as it leads to the potential deselection of normal embryos for transfer. Moreover, a proportion of uniform aneuploid embryos may be inaccurately categorized as high-level mosaic, with a consequent negative outcome (i.e. miscarriage) when inadvertently selected for transfer. Clinical outcomes following PGT-A are maximized when a 50% threshold is employed as it offers the most accurate diagnostic approach., Study Funding/competing Interest(s): The study was supported by Igenomix. The authors not employed by Igenomix have no conflicts of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform?, Summary Answer: The proposed integrated approach accurately assesses ploidy level and the most common pathogenic microdeletions causative of genomic disorders, expanding the clinical utility of PGT., What Is Known Already: Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date., Study Design, Size, Duration: This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss., Participants/materials, Setting, Methods: The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD., Main Results and the Role of Chance: In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1-100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5-99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5-4.5%) among euploid embryos., Limitations, Reasons for Caution: Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay., Wider Implications of the Findings: This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos., Study Funding/competing Interest(s): No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: What is the origin and composition of cell-free DNA in human embryo spent culture media?, Summary Answer: Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos., What Is Known Already: In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative., Study Design, Size, Duration: This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types., Participants/materials, Setting, Methods: Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes., Main Results and the Role of Chance: We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result., Limitations, Reasons for Caution: This study was limited by the sample size and the number of cells analyzed by FISH., Wider Implications of the Findings: This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos., Study Funding/ Competing Interest: This work was funded by Igenomix S.L. There are no competing interests.

Background: Ovarian stimulation regimens for in vitro fertilization seem to have a deleterious effect on oocyte quality and embryo aneuploidy in a dose-dependent manner. This study aims to test the influence of gonadotrophin doses on embryo aneuploidy rates., Methods: A total of 32 young oocyte donors with a high response to ovarian stimulation, were included in the study. Two subsequent stimulation treatments were performed in each donor: first, a standard dose cycle using a 225 IU starting dose of recombinant FSH (r-FSH) and secondly, a reduced dose cycle with a starting dose of 150 IU r-FSH. In both cycles, GnRH agonist co-treatment was used for down-regulation. Ovarian response, embryo development and aneuploidy for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were the main outcomes of the study., Results: A total of 22 donors completed both treatments with different gonadotrophin doses. In the remaining 10 donors, the reduced dose cycle was cancelled due to low ovarian response. In those donors who completed both regimens, significant increases in rates of fertilization and chromosomally normal blastocysts were observed in the reduced dose cycle. No differences were observed in pregnancy and implantation rates in recipients who received oocytes from standard and reduced doses cycles., Conclusions: Despite the limited numbers in our study, we can conclude that in high responder donors, a decrease in the gonadotrophin dose could improve fertilization rates and embryo quality. However, due to the reduced oocyte numbers with lower doses, a similar reproductive outcome in terms of live births would be expected.

Background: In our routine programme of preimplantation genetic aneuploidy screening (PGS) by fluorescence in situ hybridization (FISH), nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) are analysed in two consecutive hybridization rounds. We also perform additional hybridization rounds for these chromosomes, using probes that bind to different loci, for non-conclusive results and for confirmation of certain aneuploidies. The aim of this study was to evaluate the impact of additional hybridization rounds on FISH accuracy., Methods: This is a retrospective analysis of our FISH data from 1000 PGS cycles performed from December 2007 to December 2008 for various indications. In addition to the hybridization rounds described above, 132 of the embryos diagnosed as chromosomally abnormal were re-analysed on Day 5., Results: A total of 2477 embryos were re-hybridized, 1496 due to non-conclusive results and 981 to confirm observed aneuploidies. After re-hybridization, 882 embryos (59%) were then diagnosed as normal, 600 embryos (40.1%) had a clear abnormality and only 14 embryos (0.9%) remained non-informative. From the 981 embryos in the latter group, 890 embryos had monosomies and, after re-hybridization 174 embryos (19.6%) were normal and 716 (80.5%) had confirmed monosomies. In contrast, re-hybridization confirmed 90 (98.9%) of the 91 observed trisomies. In addition, Day-5 re-analysis of abnormal embryos showed a higher rate of concordant diagnosis between Day 3 and Day 5 when re-hybridizations had been included on Day-3 (95 versus 82.7%; P= 0.0443), especially for the confirmation of monosomies (82.8 versus 61.0%; P = 0.0087)., Conclusions: Our data indicate that additional hybridization rounds improve the accuracy of the diagnosis, increasing the number of chromosomally normal embryos available for transfer. Re-hybridization with additional probes as a standard approach to PGS could enhance the potential benefits of the technique.

Background: The role of thrombophilia and thyroid autoimmunity in unexplained infertility (UI), implantation failure (IF) and recurrent spontaneous abortion (RSA) is controversial and poorly understood., Methods: From March, 2004 to January, 2007, 119 women were prospectively included: 32 oocyte donors, 31 patients with UI, 26 with IF and 30 with RSA. The IF and RSA groups presented normal preimplantation genetic screening. Protein C, protein S, antithrombin III, lupus anticoagulant, activated protein C resistance (APCR), immunoglobulin M and G anticardiolipin antibodies, homocystine, Factor V Leiden, prothrombin G20210A mutation, methylentetrahydrofolate reductase C677T mutation, thyroid-stimulating hormone (TSH), free thyroxine, anti-thyroid peroxidase (TPO) and anti-thyroglobulin (TG) antibodies were assessed., Results: The prevalence of thrombophilia was high and similar among groups. In the IF group, the prevalence of APCR (15.4%), lupus anticoagulant (11.5%) and combined thrombophilia (19.2%) was higher, but not significantly different, than the other three groups. The prevalence of thyroid autoimmunity in women with IF (anti-TPO antibodies, P = 0.009; anti-TPO plus anti-TG antibodies,P = 0.04) and UI (anti-TPO, P = 0.002; anti-TG, P = 0.019; anti-TPO plus anti-TG antibodies, P = 0.005) was significantly increased in comparison to those with RSA. There was also a trend towards a higher prevalence of thyroid autoimmunity in the UI and IF groups than in the control group. TSH and free thyroxine levels all remained within a normal range., Conclusions: When embryo aneuploidy is ruled out, thrombophilia could constitute an etiologic factor in IF. Furthermore, thyroid autoimmunity is strongly related to UI and IF.

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age. The aim of the present study was to investigate the role of CALPAIN-5 (CAPN5) gene in PCOS susceptibility., Methods: We analysed four intronic polymorphisms of the CAPN5 gene in 148 well-characterized women with PCOS and 606 unrelated controls. We performed a case-control study and an intracohort analysis of clinical characteristics associated with PCOS., Results: Analysis of haplotypes distribution between PCOS population compared to controls showed a strong deviation (P = 0.00029). The haplotypes GGCA and GGTG were overrepresented in PCOS patients (P = 0.009 and P = 0.001, respectively). In addition, we identified several CAPN5 haplotypes associated with phenotypic differences observed between PCOS patients, such as the presence of obesity (P = 0.02), cardiovascular complications (P = 0.02), familial antecedents of obesity (P = 0.003) and of hypertension (P = 0.007) and type 2 diabetes mellitus aggregation (P = 0.04)., Conclusions: These results suggest a role of CAPN5 gene in PCOS susceptibility in humans. Moreover, novel candidate risk alleles have been identified, within CAPN5 gene, which could be associated with important phenotypic and prognosis differences observed in PCOS patients.

Background: An increased incidence of numerical chromosomal abnormalities has been reported in the ejaculated spermatozoa of infertile patients. However, there are few cytogenetic studies of testicular and epididymal spermatozoa, and their results are still controversial., Methods: Fluorescence in-situ hybridization (FISH) analysis of chromosomes 13, 18, 21, X and Y was performed on seven testicular samples and two epididymal samples from patients with obstructive azoospermia (OA), and on 13 testicular samples from patients with non-obstructive azoospermia (NOA). Five ejaculated sperm samples from normozoospermic fertile donors were evaluated as a control group., Results: Both epididymal sperm samples showed normal FISH results for the parameters analysed when compared with those of the control group. FISH results were abnormal in 29% (two of seven) of testicular samples from OA patients and in 54% (seven of 13) of those from NOA patients, although this difference was not statistically significant. Testicular samples from OA patients showed a significant increase of disomy for sex chromosomes (P<0.01), whereas NOA patients displayed significantly higher rates of diploidy (P<0.0001) and disomy for chromosomes 13 (P<0.0001), 21 (P<0.001) and sex chromosomes (P<0.0001) than the control group., Conclusions: Testicular spermatozoa from azoospermic patients present increased rates of chromosomal abnormalities, mainly of the sex chromosomes, which are particularly high in NOA patients.

Background: Pronuclear (PN) zygote morphology has been proposed as a useful tool for selecting the best embryos for transfer., Methods: PN morphology was recorded in 888 zygotes and classified according to similar/different PN size [groups A (n = 816) and B (n = 72)] and to the number, distribution and synchrony of nucleolar precursor bodies (NPB): subgroup I, pronuclei with 3-4 polarized NPB; subgroup II, 5-7 synchronic polarized NPB or 7-10 NPB distributed randomly; and subgroup III, morphologies other than those of groups I or II. Embryo development and chromosomal abnormalities were evaluated for each PN pattern., Results: In patients aged 37 years, statistical differences among groups were not observed., Conclusions: In patients aged 37 years, this correlation does not exist.

Background: Chromosomal abnormalities are an important cause of spontaneous abortion and recurrent miscarriage (RM). Therefore, we have analysed the incidence of chromosomal abnormalities and embryo development in patients with RM., Methods: Preimplantation genetic diagnosis (PGD) was performed on 71 couples with RM and 28 couples undergoing PGD for sex-linked diseases (control group). Chromosomes 13, 16, 18, 21, 22, X and Y were analysed by fluorescence in-situ hybridization., Results: The implantation rate in RM patients was 28% and three patients (13%) miscarried. The percentage of abnormal embryos was significantly increased (P < 0.0001) in RM patients compared with controls (70.7 versus 45.1%). All of the embryos were abnormal in 19 cycles (22.1%) and repeated PGD cycles yielded similar rates of chromosomal abnormalities in 14 couples. Anomalies for chromosomes 16 and 22 were significantly higher (P < 0.01) in RM cases. In the RM population, euploid embryos reached the blastocyst stage more frequently than abnormal embryos (61.7 versus 24.9%; P < 0.0001)., Conclusions: RM is associated with a higher incidence of chromosomally abnormal embryos, of which some are able to develop to the blastocyst stage. IVF plus PGD is an important step in the management of these couples, but the technique has to move towards a full chromosome analysis.

Background: An increased incidence of chromosome abnormalities has been reported in sperm samples of many infertile men by fluorescence in-situ hybridization (FISH)., Methods: Sperm aneuploidy and diploidy rates for chromosomes 13, 18, 21, X and Y were evaluated in 63 patients with normal karyotypes using dual and triple-colour FISH techniques. Indications for sperm FISH analysis were: recurrent miscarriages of unknown aetiology (RM, n = 40), repeated implantation failure after intracytoplasmic sperm injection (ICSI) (IF, n = 19), previous Down's syndrome pregnancies (n = 3), and meiotic abnormalities (MA, n = 1). Nine healthy normozoospermic donors were also evaluated as a control group., Results: A significant increase in the incidence of sex chromosome disomies was found in the RM, IF and MA groups. Oligoasthenoteratozoospermic patients (n = 21) showed significantly higher rates of diploidy and disomies for sex chromosomes and chromosomes 18 and 21 than normozoospermic patients (n = 14). Thirty-one patients with normal and seven with abnormal FISH results had undergone several ICSI treatments (108 and 23 cycles respectively). Couples with abnormal sperm FISH results showed decreased pregnancy and implantation rates and increased miscarriage rates., Conclusions: Patients with a clinical background of recurrent miscarriages of unknown aetiology or implantation failure after ICSI are at risk of showing sperm chromosomal abnormalities, the incidence of which is higher in oligoasthenoteratozoospermic patients.

Attempts to improve the outcome in IVF and related techniques has drawn our attention to the development of culture systems that can grow embryos up to the blastocyst stage. We have developed a co-culture system with autologous human endometrial epithelial cells (EEC) that retained many features of the endometrium. In this review, we analyse our experience over the last 4 years; in particular, we address the question of whether the system would be safe and useful in cases of IVF and oocyte donation with previous implantation failure, and which factors may contribute to the failure of human embryos to develop in vitro up to the blastocyst stage. In all, 168 IVF cycles were carried out in 127 patients with 3.8+/-0.2 previous implantation failure, and 80 cycles in 57 women having oocyte donation with 3.0+/-0.2 previous implantation failure. In 168 IVF cycles, a 48.9% blastocyst formation rate was recorded; 2.3+/-0.1 blastocysts were transferred per cycle and 30 clinical pregnancies obtained (11.9% implantation and 19.6% pregnancy rates). A total of 20 IVF and 15 oocyte donation patients with three previous implantation failures in whom a day 2 embryo transfer was performed were the controls. In 88% of oocyte donation cycles, a 61.0% blastocyst formation rate was observed; 2.3+/-0.1 blastocysts were transferred per cycle and resulted in 38 clinical pregnancies (32.7% implantation and 54.3% pregnancy rates). In all, 15 cycles were cancelled (9%). In oocyte donation patients with implantation failure undergoing day 2 embryo transfer, implantation and pregnancy rates were significantly lower (4.5 and 13.3%; P < 0.01) than with co-culture; however, in IVF patients with implantation failure with day 2 transfer, results (10.7 and 35%) were similar to those with co-culture. A second question addressed was whether chromosomal abnormalities may contribute to the failure of human embryos to develop in vitro. We observed the performance of human embryos from our preimplantation diagnosis programme, which were biopsied and subsequently cultured in EEC before transfer. Out of 68 chromosomally normal embryos, 37 reached the blastocyst stage (54.4%) compared with 35 out of 104 abnormal embryos (33.6%). The present study demonstrates that co-culture of human embryos with EEC and blastocyst transfer is safe, effective, and may be a new approach to improve implantation in patients with implantation failure undergoing oocyte donation, but not in IVF patients. The system shows that abnormal embryos can also grow to the blastocyst stage, although to a lower rate. Prospective randomized studies are needed to confirm the preliminary conclusion that co-culture is an acceptable system to select good quality embryos, and the endometrium is a limiting factor in implantation that needs to be carefully managed.

Zygotes morphologically classified as tripronuclear (3PN) after intracytoplasmic sperm injection (ICSI), which are thought to be digynic in their origin, were studied by fluorescent in-situ hybridization (FISH). FISH results allowed us to assess the suspected ploidy after morphological evaluation of the zygote and to determine the origin of the third pronuclei. Our results show that, firstly, 36% of those zygotes classified as 3PN following their morphological evaluation were, in fact, diploid, and secondly, the main cause for triploidy after ICSI is the non-extrusion of the second polar body.

To assess the impact of endometriosis on intracytoplasmic sperm injection (ICSI) outcome, we have retrospectively evaluated 980 ICSI cycles, comparing the results of women with and without endometriosis. A total of 101 cycles was identified in which various degrees of endometriosis were involved, and in the remaining 879 cycles, male infertility was the only cause of infertility. Ejaculated spermatozoa were microinjected in all cycles. There was a significant reduction (P = 0.004) in the number of oocytes retrieved from women with endometriosis as compared to those without endometriosis. However, there were no significant differences in either fertilization or pregnancy and implantation rates between women with or without endometriosis. We conclude that the presence of endometriosis in patients undergoing ICSI because of severe male infertility does not affect fertilization, pregnancy and implantation rates, although significantly fewer oocytes are retrieved from patients with endometriosis.

In 25 patients (14 suffering from obstructive azoospermia, six from non-obstructive azoospermia, three from asthenoazoospermia and two from absence of ejaculation) spermatozoa were extracted from testicular biopsies. Intracytoplasmic sperm injection (ICSI) with fresh testicular spermatozoa was performed in 18 cases; spermatozoa in excess were cryopreserved in pills. No pregnancies were achieved. In the remaining seven patients, testicular spermatozoa were retrieved and cryopreserved during a diagnostic testicular biopsy. After thawing, sperm motility was assessed in 17 cases (68%), and 18 ICSI with cryopreserved testicular spermatozoa were performed. The mean two-pronuclear (2PN) fertilization rate was 59%, the mean cleavage rate was 92%, and six clinical pregnancies were achieved, all of them still ongoing (pregnancy rate 33%). A comparison of the results of ICSI carried out with fresh or cryopreserved testicular spermatozoa showed that the mean 2PN fertilization rates per cycle (53 compared with 55%), mean cleavage rates per cycle (99 compared with 96%) and embryo quality were not significantly different. In conclusion, cryopreservation of testicular spermatozoa is feasible, even in patients with non-obstructive azoospermia, and the results of ICSI with frozen-thawed testicular spermatozoa are similar to those obtained using fresh testicular spermatozoa. Cryopreservation of testicular spermatozoa may avoid repetition of testicular biopsies to retrieve spermatozoa for successive ICSI cycles in patients in whom the only source of motile spermatozoa is the testicle.

In patients with obstructive azoospermia in whom standard microsurgical procedures fail or are unfeasible, the only source of spermatozoa is the testicle. In addition, in some azoospermic patients with severe spermatogenic failure, a few spermatozoa may be present in testicular biopsy specimens despite high serum follicle stimulating hormone concentrations. In all these cases, intracytoplasmic sperm injection (ICSI) with testicular biopsy-extracted spermatozoa may offer the chance of pregnancy. To assess the efficacy of this procedure, we compared the results of two series of ICSI cycles performed during the same time period: 21 cycles using testicular biopsy-extracted spermatozoa and 83 cycles using ejaculated spermatozoa. Mean fertilization rates (59% with testicular and 68% with ejaculated spermatozoa), mean cleavage rates (93% with testicular and 90% with ejaculated spermatozoa), embryo quality (77% good quality embryos in the testicular sperm group and 77% in the ejaculated sperm group) and clinical pregnancy rates (36.8% in the testicular sperm group and 28% in the ejaculated sperm group) were not significantly different in both groups. We conclude that high fertilization, cleavage and pregnancy rates can be achieved with intracytoplasmic testicular sperm injection, reaching levels comparable with those of ICSI using ejaculated spermatozoa.