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STUDY QUESTION Is the presence of DNA in the blastocoel fluid (BF) of expanded blastocysts, assessed by whole genome amplification (WGA), associated with the clinical outcome at the first transfer? SUMMARY ANSWER At the first transfer, blastocysts with negative BF-WGA have more chance to implant and to develop to term than those with positive BF-WGA results, both in preimplantation genetic testing for aneuploidies (PGT-A) cycles (where only euploid blastocysts resulting from the chromosomal analysis of trophectoderm (TE) biopsies were transferred) and in IVF/ICSI conventional cycles. WHAT IS KNOWN ALREADY Retrospective studies conducted in patients undergoing PGT-A have shown that the incidence of negative BF-WGA was significantly higher in TE-euploid blastocysts than in TE-aneuploid blastocysts. In addition, after the transfer of TE-euploid blastocysts, the ongoing clinical pregnancy rate was significantly higher in the group with negative BF-WGA compared with those with positive BF-WGA. STUDY DESIGN, SIZE, DURATION A prospective cohort study including 102 consecutive PGT-A patients (Group 1) and 88 consecutive conventional IVF/ICSI patients (Group 2), was conducted between January 2019 and December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS In both groups, BFs were collected from expanded blastocysts of high grade and processed for WGA. DNA amplification was evaluated by agarose gel electrophoresis for the presence (positive BF-WGA) or absence (negative BF-WGA) of a band. Directly after the BF retrieval, blastocysts from Group 1 underwent TE biopsy and vitrification. In Group 2, blastocysts were vitrified immediately after BF collection. In Group 1, only euploid blastocysts were considered for transfer according to the results of TE biopsies. In both groups, the selection of the blastocyst to be transferred was based on BF-WGA results giving priority, if available, to those with negative amplification. The primary outcome investigated was the live birth rate (LBR) at the first transfer. The main variable under investigation was the negative BF-WGA and results were corrected for confounders (maternal and paternal age, number of retrieved oocytes, male factor) by multiple logistic regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE In Group 1, 60 patients transferred negative BF-WGA blastocysts and 42 positive BF-WGA blastocysts, and the LBR at the first transfer was 53.3% and 26.2%, respectively (P = 0.0081). After testing for selected confounders in a multiple logistic analysis, the transfer of blastocysts with negative BF-WGA resulted in an odds ratio of (OR) 3.52 (95% CI: 1.48–8.88, P = 0.0057) compared to transfer of positive BF-WGA blastocysts. In Group 2, at the first transfer 30 deliveries resulted from blastocysts with negative BF-WGA (48.4%) and three from the transfer of positive BF-WGA blastocysts in 26 patients (11.5%; P = 0.0014). Multiple logistic analysis indicated that the transfer of blastocysts with negative BF-WGA resulted in an OR 6.89 (95% CI: 1.98–32.95, P = 0.0056) compared to transfer of positive BF-WGA blastocysts. The LBR per transfer and the cumulative LBR per patient showed the same trend. LIMITATIONS, REASONS FOR CAUTION The study was performed in a single center. WIDER IMPLICATIONS OF THE FINDINGS The data from this study highlight the heterogeneity of blastocysts of similar morphology, even in those classified as euploid by TE analysis. Failure to detect DNA in BFs after WGA is associated with a significantly higher LBR at the first embryo transfer as well as per transfer and per patient. The processing of the BF by WGA is an easy and cost-effective tool that could become a valuable option to offer patients the highest chances of term pregnancy in the shortest time possible. STUDY FUNDING/COMPETING INTEREST(S) The study received no funding from external sources. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

RESEARCH QUESTION : Is the efficacy of imported vitrified oocytes donation affected by the cryobank of origin? DESIGN : Longitudinal cohort study including 249 completed oocyte warming cycles from 200 recipients (January 2016-July 2020). No severe male factor was included. Vitrified oocytes were provided by three Spanish cryobanks. The primary outcome was the cumulative live birth-delivery rate (cLBR) per completed oocyte warming cycle. RESULTS : After warming 1535 oocytes, 1244 survived (81.0%) and 945 fertilized (76.0%) with an embryo utilization rate of 65.3%. The overall cLBR per completed cycle was 47.0%, but it was lower in cryobank 1 (31.2%) in comparison to cryobank 2 (56.0%, p=0.0010) and 3 (50.8%, p=0.0241). The multivariate logistic regression analysis identified the survival of ≥4 oocytes as the strongest predictor for delivery (p=0.0282). Only 202 of the 249 oocyte warming cycles had ≥4 survived oocytes in a proportion that was significantly lower in cryobank 1 versus cryobank 2 (70.1% vs. 89.0%, p=0.0020), whereas the comparison with cryobank 3 (81.0%) was not significant. In the 202 oocyte warming cycles, the cLBR in cryobank 1 (37.0%) was lower in comparison to cryobank 2 (58.8%, p=0.0115) and cryobank 3 (60.8%, p=0.0019) suggesting a reduced viability also in oocytes from cryobank 1 that survived warming. CONCLUSIONS : There are differences in the efficacy of the imported vitrified oocytes in relation to the cryobank of origin. Each center needs to evaluate the results internally when starting a collaboration with an oocyte cryobank to put in place the necessary measures to maximize treatments’ efficacy.

The incorporation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing into patient care algorithms has been proposed to mitigate risk. However, the two main professional societies for human reproduction (ESHRE and ASRM) appear divergent on their clinical utility and whether they should be adopted. In this opinion paper, we review the currently available tests and discuss the strengths and weaknesses of the proposed clinical care pathways. Nucleic acid amplification tests are the cornerstone of SARS-CoV-2 testing but test results are largely influenced by viral load, sample site, specimen collection method, and specimen shipment technique, such that a negative result in a symptomatic patient cannot be relied upon. Serological assays for SARS-CoV-2 antibodies exhibit a temporal increase in sensitivity and specificity after symptom onset irrespective of the assay used, with sensitivity estimates ranging from 0 to 50% with the first 3 days of symptoms, to 83 to 88% at 10 days, increasing to almost 100% at ≥ 14 days. These inherent constraints in diagnostics would suggest that at present there is inadequate evidence to utilize SARS-CoV-2 testing to stratify fertility patients and reliably inform clinical decision-making. The failure to appreciate the characteristics and limitations of the diagnostic tests may lead to disastrous consequences for the patient and the multidisciplinary team looking after them.

STUDY QUESTION What has the ESHRE programme ‘ESHRE Certification for Clinical Embryologists’ achieved after 10 years? SUMMARY ANSWER The post-exam analysis showed a pass rate of 60% for Clinical and 50% for Senior Clinical Embryologists and a high level of internal consistency of all exams, leading to a total of 773 certified Clinical and 493 Senior Clinical Embryologists over the decade. WHAT IS KNOWN ALREADY In an ESHRE survey on the educational and professional status of Clinical Embryology in Europe, it was found that education of laboratory personnel working in the field of assisted reproduction is highly variable between countries. In 2008, ESHRE introduced a programme, curriculum and certification in the field of Clinical Embryology. Knowledge gained by postgraduate study of recommended literature, following a clear curriculum, is verified by a written two-level exam for obtaining a certificate for Clinical (basic) or Senior Clinical (advanced) Embryologists. With a total of 1266 certificates awarded over a period of 10 years and recognition by the Union Européenne des Médecins Spécialistes and their Council for European Specialists Medical Assessment, the ESHRE Clinical Embryology exams have become an internationally recognized educational standard in the field of Clinical Embryology. STUDY DESIGN, SIZE, DURATION A retrospective analysis of all applications for ESHRE Clinical (2009–2018) and Senior Clinical Embryologist Certification (2008–2018) and exam results of the first decade was carried out by the Steering Committee for Clinical Embryologist Certification. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 2894 applications for ESHRE Certification for Clinical Embryologists and the results of 10 exams for the Clinical (1478 candidates) and 11 exams for Senior Clinical (987 candidates) levels were analysed. A detailed post-exam retrospective analysis was performed regarding difficulty, discrimination and reliability levels of 1600 multiple-choice questions (MCQs) with a single best answer among four options, from eight different curriculum topics (Basic cell biology, Genetics, Developmental biology, Female reproduction, Male reproduction, IVF laboratory, Cryopreservation and Laboratory management), representing the core theoretical knowledge of Clinical Embryology. Difficulty levels of the MCQs were subsequently compared regarding each topic and each yearly exam. The participation and success rates in the ESHRE Clinical Embryology exams were also assessed in terms of the educational and geographic backgrounds of candidates. MAIN RESULTS AND THE ROLE OF CHANCE Over the 10 years studied, the mean pass rate for the Clinical Embryologist exam was 60% (range 41–86%), and for the Senior Clinical Embryologist exam was 50% (range 34–81%). On average, 63% European candidates and 35% non-European candidates passed the Clinical Embryologist exam, while 52% European candidates and 31% non-European candidates passed the Senior Clinical Embryologist exam. The candidates’ educational level impacted on the success of the Clinical Embryologist exam but not of the Senior Clinical Embryologist exam. The mean difficulty indices by study topic showed that in the period of 10 years, there were no statistically significant differences between topics, for either the Clinical or Senior Clinical Embryologist exams. However, the overall exam difficulty varied between years. Reassuringly, the exam MCQ discrimination and reliability indices always showed a high level of internal consistency in all exams. LIMITATIONS, REASONS FOR CAUTION Some data from the initial ESHRE certification programme were not obtained electronically, in particular data for education, implying tables and figures reflect the specified valid data periods. Several countries exhibit different study profiles for those working in ART laboratories, such that laboratory technicians/technologists predominate in some countries, while in others only biologists and medical doctors are allowed to work with human embryos. Such differences could consequently affect the exam performance of candidates from specific countries. WIDER IMPLICATIONS OF THE FINDINGS The ESHRE exams on Clinical Embryology are the most widely, internationally accepted tests of knowledge in the rapidly growing area of human reproduction. Clinical Embryology is increasingly recognized as a specific discipline for scientific staff who are collaborating closely with clinicians in managing human infertility through medically assisted reproduction. The analysis of the first 10 years of application of a two-level exam for Clinical Embryology shows a consistent high quality and reliability of the exam and MCQs used. These results represent an important follow-up of the quality of the ESHRE Certification programme for Clinical Embryologists, and convincingly position Clinical Embryology in the wider group of health disciplines that are harmonized through professional bodies such as ESHRE and European Board & College of Obstetrics and Gynaecology. The exams provide a clear step towards the increasing professional recognition and establishment of Clinical Embryology within health systems at both European and international level. STUDY FUNDING/COMPETING INTEREST(S) No competing interest. All costs of the Steering Committee meetings were covered by ESHRE.

This is a pilot study performed in a private IVF unit. The objective of the study was to investigate whether luteal support is required in IVF cycles after mild stimulation with clomiphene citrate and low FSH doses. The study included 15 patients with good prognosis (defined as ≤38 years old with normal ovarian reserve and normovulatory cycles, body mass index

Study question Is it important that end-users know the composition of human embryo culture media? Summary answer We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. What is known already Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. Study design, size, duration A review of the literature was carried out. Participants/materials, setting, methods Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. Main results and the role of chance The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. Limitations, reasons for caution There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Wider implications of the findings Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the composition of embryo culture media. Study funding/competing interests This work was funded by The European Society for Human Reproduction and Embryology. No competing interests to declare.

Objective To investigate blastocysts, defined as euploid and aneuploid by trophectoderm (TE) cell analysis, for the presence of DNA in the blastocoelic fluid (BF) detected by whole-genomic amplification (WGA); and to correlate the presence of DNA in BF with the clinical outcome after the transfer of TE-euploid blastocysts. Design Retrospective study. Setting In vitro fertilization unit. Patient(s) This study included 91 patients performing preimplantation genetic testing for aneuploidy on TE cells from January 2015 to December 2017. In the case of ET, only single blastocyst transfers were performed. Intervention(s) Blastocoelic fluids and TE cells were retrieved from 256 blastocysts before vitrification. All blastocysts were diagnosed by array-comparative genomic hybridization (a-CGH) on TE cells. Amplification and a-CGH of DNA from BFs was performed at a later time after TE biopsy and ET. Main Outcome Measure(s) Whole-genomic amplification of BFs, evaluation of the chromosome condition in BFs and TE cells, and correlation of BF results with the clinical outcome of TE-euploid transferred blastocysts. Result(s) The incidence of amplification after WGA was significantly lower in BFs from TE-euploid blastocysts (n = 32, 45%) when compared with the aneuploid ones (n = 150, 81%), resulting in 182 BFs with successful DNA amplification. When submitted to a-CGH, informative results were obtained from 172 BFs. Comparison of these results with those from the corresponding TE cells gave a ploidy concordance of 93.6% and a mean number of aneuploid events per sample that was higher in BFs than in TE cells (2.0 vs. 1.4, respectively). After the transfer of 53 TE-euploid blastocysts, the clinical pregnancy rate was 77% in the group with BF-failed amplification, and 37% after BF-successful amplification. The same trend was found for the ongoing pregnancy rate (68% vs. 31.5%, respectively). Conclusion(s) The presence of DNA in BFs detected by WGA is correlated with the blastocyst ploidy condition defined by TE cell biopsy and with the implantation potential of TE-euploid blastocysts. These findings could have a clinical implication for the selection of the most viable embryo for transfer because, after submitting BFs to WGA, priority would be given to TE-euploid blastocysts with BF-failed amplification. Similarly, BF-failed amplification could be an additional selection criterion to prioritize embryos for transfer even in conventional IVF cycles with blastocysts that were vitrified after BF aspiration.

Birefringence in sperm heads reflects an organized and very compacted texture, indicating nuclear and acrosomal structural normality. This study performed a direct analysis of the acrosome integrity in single spermatozoa to verify whether a pattern of total or partial head birefringence reflected the acrosome status. The morphology in fresh samples was assessed according to World Health Organization criteria while the characteristics of birefringence were evaluated by polarized light. Acrosome integrity was evaluated by fluorescein isothiocyanate Pisum sativum agglutinin that binds selectively to the acrosome content. According to the results, a reacted acrosome was present in 96% of spermatozoa with partial birefringence and only in 35% of those with totally birefringent heads. A great proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P Human sperm cells are naturally birefringent when observed in a polarizing light due to the presence of longitudinally oriented protein filaments. The detection of birefringence in sperm heads reflects an organized and very compacted texture that indicates nuclear and acrosomal structural normality. Basically, three types of head birefringence have been observed: total, partial (localized in the post acrosomal region) and abnormal (absent or irregular). Data from electron microscopy has suggested a correlation between acrosome integrity and birefringence patterns. The aim of this study was to verify this hypothesis by direct analysis of the acrosome status in single spermatozoa. The morphology in fresh samples was assessed according to World Health Organization criteria while the characteristics of birefringence were evaluated by polarized light. Acrosome integrity was evaluated by fluorescein isothiocyanate Pisum sativum agglutinin that binds selectively to the acrosome content. According to the results, 96% of spermatozoa with partial birefringence had a reacted acrosome, while 65% of totally birefringent heads had an intact acrosome. A large proportion of sperm cells with normal morphology showed total birefringence both in the presence (59%) or in the absence of motility (45%; P

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes. European Journal of Human Genetics (2012) 20, 742-747; doi: 10.1038/ejhg.2011.272; published online 8 February 2012

There are many examples in assisted reproduction technology, where new technology and methods have been introduced into the clinical setting without appropriate development and evidence-based medicine to show that the procedure is safe and beneficial to the patient. Examples include preimplantation genetic screening, assisted hatching, in vitro maturation, blastocyst transfer and vitrification. Changes to culture media composition, stimulation regimes and laboratory protocols are also often established internationally without ad- equate validation. More recently, novel equipment that needs to be validated before it enters routine clinical use is being developed for IVF. With technologies such as producing gametes from stem cells around the corner, it is vital to ensure that the necessary research and de- velopment is conducted before bringing new techniques into clinical practice. Ideally, this should include preliminary work on animal models, such as mice/rats/rabbits/larger mammals, followed by studies on human embryos donated for research and finally well-designed RCTs with a follow up of all children born from the procedure. If such preliminary studies are not performed and published, it is possible that technology bringing no clinical benefit or leading to adverse health outcomes in the children born by these practices may be introduced. All IVF clinics need to consider the safety and efficacy of new technologies before introducing them.

Meiotic spindle (MS) assembly in human oocytes is a dynamic process that can be visualized by computer-assisted microscopy. At extrusion of the first polar body a spindle bridge is detected until the completion of telophase I and its reformation requires approximately 1h. This study analysed 396 oocytes from 112 cycles for fertilization and cleavage according to MS detection at two examinations, 39 and 41h post-human chorionic gonadotrophin (HCG). All cycles had at least one injected oocyte lacking a visible MS at intracytoplasmic sperm injection (41h post-HCG). To evaluate the results, oocytes were divided according to the presence (group A) or absence at both observations (group B) of the MS. Compared with group A, group B oocytes had lower normal fertilization rates, higher incidence of three pronuclei and two pronuclei in early dissolution and lower development to blastocyst. Some group A oocytes showed a late MS formation (not visualized at 39h but at 41h) and their performance was similar to that of the oocytes with a MS visible at both time points. Although some implantations occurred in group B, these findings suggest that prolonged MS non-detection could be a marker of reduced oocyte competence. Birefringence is an optical property associated with molecularly ordered structures, such as microtubules and other cytoskeletal components, that is visualized by polarization microscopy. As birefringence has been associated with cell integrity and viability, its non-invasive nature encouraged the clinical application. The oocyte's meiotic spindle (MS) is a birefringent structure controlling the correct chromosome alignment in the metaphase plate. According to recent publications, the MS visualization in mature oocytes is related to fertilization and cleavage, but a correlation with the clinical outcome is not so evident. As MS formation is a dynamic process, this study analysed the MS by sequential birefringence imaging at 39 and 41h post human chorionic gonadotrophin (just before intracytoplasmic sperm injection) in a homogenous group of patients to verify whether this parameter is associated with following development. All cycles had at least one oocyte lacking a visible MS. To evaluate the results, oocytes were divided according to the presence (group A) or prolonged absence at both observations (group B) of the MS. Compared with group A, group B oocytes had lower normal fertilization rates, higher incidence of three pronuclei and of two pronuclei in early dissolution and lower development to blastocyst. Some group A oocytes showed late MS formation (not visualized at 39h but at 41h) and their performance was similar to that of oocytes with a MS visible at both imaging. Although some implantations occurred in group B, these findings suggest that prolonged MS non-detection could be a marker of reduced oocyte competence.

To identify factors that might affect the clinical outcome of oocyte slow freezing.Retrospective study.Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy.Patients with spare metaphase II cryopreserved oocytes performing 371 thawing cycles.Oocytes were cryopreserved by slow freezing40 hours after hCG administration (group A) andor=40 hours after hCG administration (group B). Thawed oocytes were inseminated by intracytoplasmic sperm injection.Clinical pregnancy, implantation, abortion, and delivery rates.Clinical pregnancy rate per thawed cycle (PR) and implantation rate (IR) were significantly higher in group A compared with group B both in young (PR: 25% vs. 9.6%; IR: 18.9% vs. 8.8%) and in older patients (PR: 25% vs. 10.1%; IR: 17.5% vs. 6.7%). In the young patient subgroup, clinical pregnancy and implantation rates with three transferred embryos were higher in group A vs. group B (PR: 72.7% vs. 25%, and IR: 36.4% vs. 12.5%, respectively). This difference was not found in the subgroup of older patients.The timing at which oocyte cryopreservation is performed and the number of transferred embryos play a key role in the clinical outcome. The suggested cut-off time for cryopreservation is between 39 and 40 hours after hCG administration.

Objective To verify whether the morphologic evaluation of zygotes and embryos derived from thawed oocytes could provide some relevant information regarding their developmental performance. Design Fertilization, zygote, and embryo morphology from sibling fresh and frozen oocytes was compared. Setting Reproductive Medicine Unit, Societa Italiana Studi Medicina della Riproduzione, Bologna, Italy. Patient(s) Two hundred thirty-four patients underwent intracytoplasmic sperm injection cycles from which 1,101 spare metaphase II oocytes were cryopreserved. Subsequently, 256 thawing cycles were performed, and 997 oocytes were thawed. Intervention(s) Intracytoplasmic sperm injection was performed on both fresh and frozen oocytes. Main Outcome Measure(s) Fertilization rates, pronuclear zygote morphology, and embryo cleavage rates. Result(s) Thawed oocytes had lower chances of being fertilized and developing into top-quality zygotes and regularly cleaving embryos when compared with sibling fresh oocytes irrespective of female age. As a result, the percentage of transferred cycles was significantly lower in frozen cycles compared with fresh cycles (79% and 93%, respectively); the proportion of transferred top-quality embryos followed the same trend. Conclusion(s) Reduced fertilization and cleavage rates in frozen cycles when compared with sibling fresh oocytes suggest that, even if surviving thawing, the process of slow freezing has a negative impact on the potential of further growth that is evident as early as the first cleavage divisions.

Objective To investigate the characteristics of birefringence in human sperm heads and apply polarization microscopy for sperm selection at intracytoplasmic sperm injection (ICSI). Design Prospective randomized study. Setting Reproductive Medicine Unit, Societa Italiana Studi Medicina della Riproduzione, Bologna, Italy. Patient(s) A total of 112 male patients had birefringent sperm selected for ICSI (study group). The clinical outcome was compared with that obtained in 119 couples who underwent a conventional ICSI cycle (control group). Intervention(s) The proportion of birefringent spermatozoa was evaluated before and after treatment in relation to the sperm sample quality. Embryo development and clinical outcome in the study group were compared with those in the controls. Main Outcome Measure(s) Proportion of birefringent sperm heads, rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. Result(s) The proportion of birefringent spermatozoa was significantly higher in normospermic samples when compared with oligoasthenoteratospermic samples with no progressive motility and testicular sperm extraction samples. Although fertilization and cleavage rates did not differ between the study and control groups, in the most severe male factor condition (oligoasthenoteratospermic with no progressive motility and testicular sperm extraction), the rates of clinical pregnancy, ongoing pregnancy, and implantation were significantly higher in the study group versus the controls. Conclusion(s) The analysis of birefringence in the sperm head could represent both a diagnostic tool and a novel method for sperm selection.

The 'ESHRE Guidelines for Good Practice in IVF Laboratories' were drawn up by the Special Interest Group (SIG) in Embryology and published in the year 2000, and since then they constitute the minimal requirements for any laboratory offering assisted reproduction techniques (ART). In the understanding that the embryologist has a responsibility for the correct and justified application of ART in the laboratory, the implementation of these guidelines requires a quality management programme to be in place that encompasses and integrates the operative units, the processes and procedures that represent the core of ART clinics. In March 2004, the European Parliament issued the Directive 2004/23/EC 'On setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells'. The Directive applies to human tissues and cells, including fresh or frozen reproductive cells for application to the human body, and is mainly concerned with increasing quality and safety through the implementation of a quality management system. Therefore, the European Society of Human Reproduction and Embryology (ESHRE) undertook a series of initiatives aiming to promote assurance of good laboratory practice and to define the concept of qualified embryologists. One ESHRE initiative was to revise the guidelines for good practice in IVF laboratories, not only in response to the need of embryologists for support and guidance in their duties, but also as a complement to the requirements issued by the Tissue and Cell Directive. The SIG in Embryology hopes that this document may assist the laboratory staff to operate according to the requirements of harmonization, implementation, inspection and certification that are now common to all European member states.

Objectives To find whether chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres of human embryos are nonrandomly localized, whether their aneuploidy affects their localization and if eventual early inactivation of chromosome X with peripheral localization is present. Methods Relative distances from the nucleus center and edge of 1198 fluorescence in situ hybridization signals in 98 human blastomeres were measured in digital images for comparison with a mathematical model of random distribution in spherical nucleus. Results Comparison with the mathematical model revealed that localization of chromosomes 13, 16, 21, 22, X and Y in normal and aneuploid blastomeres and that of chromosome 18 in normal blastomeres was not significantly different from random distribution. Similarly, chromosome X in blastomeres with more than one X did not appear to have a preferential localization. Only chromosome 18 in aneuploid blastomeres was differently distributed (p < 0.0001) with a shift to the nuclear periphery (p = < 0.0001). Conclusions Peripheral localization of chromosome 18 in aneuploid blastomeres is related to embryo aneuploidy. Conversely, a peripheral localization of the inactive X chromosome was not found in blastomeres from 3-4 day old embryos. These results open the possibility to improve embryo selection after pre-implantation diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.

Objective: To investigate chromosomal errors detected by first polar body (PB) biopsy in relation to the nuclear maturity of the oocytes. Design: Retrospective study. Setting: Reproductive medicine unit. Patient(s): Eighty-seven cycles were examined by PB biopsy for aneuploidy. Indications were maternal age 38 years (49 cycles), repeated IVF failures (22 cycles), and others (16 cycles). Intervention(s): First polar bodies were analyzed for the chromosomes 13, 16, 18, 21, and 22 in both in vivo and in vitro matured oocytes. Euploid oocytes were inseminated by intracytoplasmic sperm injection. Main Outcome Measure(s): Chromosomal status of the analyzed oocytes, development after intracytoplasmic sperm injection, pregnancy, and implantation rates. Result(s): In in vitro matured oocytes, the proportion of chromosomal abnormalities was higher than in in vivo matured oocytes (70% vs. 54%, P.005), with complex abnormalities being the prevailing defect (62% vs. 40%, P.001). Conversely, the presence of an extra chromatid or the lack of a chromatid was more frequent in in vivo than in in vitro matured oocytes (55% vs. 34%, P.001). Conclusion(s): The low viability of in vitro matured oocytes from stimulated cycles could be related to a significantly higher proportion of chromosomal abnormalities compared with in vivo matured oocytes. Complex abnormalities, involving two or more chromosomes, gave the strongest contribution to the detected increase. (Fertil Steril 2006;86:629‐35. ©2006 by American Society for Reproductive Medicine.)

BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced number of sperm cells was designed. METHODS: A severe male factor condition was the main cause of infertility for 38 couples: 27 were oligoasthenoteratospermic (OAT) and 11 with non-obstructive azoospermia underwent testicular sperm extraction (TESE). A two-round fluorescence in situ hybridization (FISH) protocol was performed with probes specific for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The recording of the position of each sperm cell at the microscope allowed diagnosis of each spermatozoon for the nine tested chromosomes. RESULTS: A mean number of 122 ± 78.5 sperm were diagnosed per patient with an incidence of total abnormalities corresponding to 13.4%. X 2 -tests for the observed frequencies and goodness-of-fit test were highly significant in all cases. A significantly higher proportion of total aneuploidy was detected in 79% of the tested samples compared to the normal population. Testicular sperm were significantly more prone to aneuploidy than ejaculated sperm. CONCLUSIONS: The designed FISH protocol for the analysis of severe OAT and TESE sperm samples is reliable, implying that the studied sample is representative of the original population. In view of the high incidence of aneuploidy in most severe OAT and TESE sperm, the FISH analysis of pathological sperm samples can be routinely performed in order to estimate the chances of the paternal contribution to aneuploidy in the resulting embryos.

The use of multiple probes in fluorescence in situ hybridization (FISH) permits the simultaneous analysis of several chromosomes in both blastomeres and spermatozoa. Preimplantation genetic diagnosis (PGD) for aneuploidy provides information on embryonic chromosomal status, enabling the selection of embryos carrying aneuploid condition. This strategy directly affects implantation, as documented for patients with a poor prognosis for pregnancy, who have the tendency to generate high proportions of chromosomally abnormal embryos. PGD for aneuploidy also has contributed information on early phases in human embryology by clarifying the molecular basis in some cases of irregular development. Multicolor FISH has also been used to study chromosomes on spermatozoa. Experimental strategies and modifications enabled the analysis of samples with a very low number of sperm cells, including samples retrieved from the genital tract or directly from the testicular tissue. The results confirmed that the incidence of aneuploidy increases proportionally with the severity of the male-factor condition. This observation suggests that, in selected cases, the paternal contribution to aneuploidy in the developing conceptus could be more relevant than expected from general data from aborted fetuses and live births.

The aim of this study was to evaluate the clinical impact of preimplantation genetic diagnosis (PGD) for aneuploidy on 193 patients who subsequently achieved 208 clinical pregnancies, in relation to their reproductive history. The 208 clinical pregnancies included in the study resulted from 1029 assisted conception cycles in combination with PGD for aneuploidy in 740 couples with a history of poor reproductive performance. According to the reproductive history of the 193 patients, 61 had previously experienced 112 pregnancies with 105 abortions and seven deliveries, corresponding to 3.6% take-home baby rate and 10.9% implantation rate. During the PGD cycle, preimplantation embryos were analysed for 5-9 chromosomes. The transfer of euploid embryos was performed in 699 cycles (68% of oocyte retrievals), generating 171 term pregnancies with 210 infants born, whereas 34 aborted spontaneously and three were ectopic, giving a take-home baby rate per pregnant patient of 88.6% and an ongoing implantation rate per pregnant patient of 53.2%. According to these data, selection made in preimplantation embryos against chromosomal abnormalities is associated with a significantly higher (P < 0.001) take-home baby rate when compared with the previous reproductive history of the parents.

This study evaluated the chromosomal condition of embryos generated by patients with an altered karyotype due to gonosomal mosaicism and the clinical outcome after preimplantation genetic diagnosis (PGD) for aneuploidy. Thirty-six patients aged 34.6 ± 3.6 years performed 54 treatment cycles and had 295 embryos diagnosed by fluorescence in-situ hybridization (FISH). Thirty-seven per cent of the embryos were chromosomally normal and generated 19 clinical pregnancies after replacement in 39 cycles. Only one pregnancy miscarried, yielding a take-home baby rate of 33.3%. Autosomal monosomy and trisomy contributed 36.1% of total abnormalities and gonosomal aneuploidy 5.9%, similar to the results detected in patients who undergo PGD for increased maternal age. Reanalysis was performed on 114 non-transferrable embryos: 41 were found to be mosaics, which were grouped in three different types, chaotic mosaics (56%), aneuploid mosaics (29%) and diploid/haploid/polyploid mosaics (15%). The incidence of aneuploid mosaics was higher than expected compared with PGD patients of the same age and resembled the condition observed in patients of advanced maternal age. These findings suggest that constitutional carriers of sex chromosome mosaicism are predisposed to autosomal mosaicism of embryos, possibly due to errors of cell division. There is an indication that this tendency is higher in female than male carriers.

To investigate the mitochondrial DNA (mtDNA) segregation in human oocytes, the level of heteroplasmy in the three products of meioses, polar bodies (PBs) and corresponding oocytes, was assessed by studying the hypervariable region I (HVRI) of the D-loop region. The DNA from 122 PBs and 51 oocytes from 16 patients was amplified by whole genome amplification (WGA). An aliquot of the WGA product was used to assess aneuploidy, and another aliquot to study mtDNA. The HVRI was amplified and sequenced with an efficiency of 75.4 and 63%, respectively, in PBs, and of 100% in oocytes. The comparison with the mtDNA sequences from blood of the individual donors showed full correspondence of polymorphisms with the matching oocytes, whilst in PBs the degree of concordance dropped to 89.6%. Haplogroups were inferred for all 16 patients. Of the 89 diagnosed PBs from the 13 patients belonging to macrohaplogroup R, 23 were euploid and 66 aneuploid. The incidence of total anomalies was significantly lower in haplogroup H (6.5%) when compared with haplogroups J and T (17.6 and 13.4% respectively; P < 0.001). In haplogroup J, hypoaneuploidy occurred more frequently than hyperaneuploidy. In the three patients belonging to haplogroup N*, 81% of PBs were aneuploid with similar rates of chromosome hypoaneuploidy and hyperaneuploidy. The presence of mtDNA base changes confined to PBs could reflect a selection mechanism against severe mtDNA mutations, while permitting a high evolution rate that could result in bioenergetic diversity. The different susceptibility to aneuploidy by some haplogroups strongly supports this hypothesis.

Objective To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies (PBs) or blastomeres. Design Prospective study. Setting In vitro fertilization unit. Patient(s) Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization on PBs (n = 12) or blastomeres (n = 5). Intervention(s) BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis. Main Outcome Measure(s) Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the results in TE cells and those predicted by the analysis done at earlier stages. Result(s) DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively. Conclusion(s) Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly predictive of the embryo ploidy condition and chromosome content. Our data confirm the relevance of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the resulting blastocyst.

Objective: To verify whether advantages can derive from the implementation of preimplantation genetic diagnosis for aneuploidy in patients with a poor prognosis of full-term pregnancy, compared with conventional treatment procedures. Design: A randomized, controlled study. Setting: Reproductive Medicine Unit of the Societa Italiana Studi Medicina della Riproduzione, Bologna, Italy. Patient(s): In a total of 262 stimulated cycles, women presented with the following poor-prognosis indications: maternal age of ≥36 years (n = 157), ≥3 previous IVF failures (n = 54), and an altered karyotype (n = 51). After giving consent, 127 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 135 controls underwent assisted zona hatching. Intervention(s): Analysis of chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 was carried out with the fluorescence in situ hybridization technique in a blastomere biopsied from day 3 embryos. Assisted zona hatching was performed on day 3 embryos from the control group. Main Outcome Measure(s): Embryo morphology and chromosomal status, number of transferred embryos, clinical pregnancies, implantation rates, and abortions. Result(s): In the study group, 717 embryos were analyzed by fluorescence in situ hybridization, and 60% were chromosomally abnormal. A mean of 2.3 ± 0.9 euploid embryos were transferred in 99 cycles, resulting in 37 clinical pregnancies (37%) and a 22.5% ongoing implantation rate. In the control group, 126 cycles were performed with 3.2 ± 1.3 embryos transferred, yielding 34 clinical pregnancies (27%) and a 10.2% ongoing implantation rate. Conclusion(s): The advantage of selecting embryos with a normal chromosome complement has an immediate impact on the ongoing implantation rate, especially in patients aged ≥38 years and carriers of an altered karyotype.

Introduction Aneuploidy is the most common chromosome abnormality in humans, and is the main genetic cause of miscarriage and congenital birth defects following both natural conception and in vitro fertilization (IVF). It is now known that the majority of chromosome errors originate in maternal meiosis I (MI), maternal age is a risk factor for most human aneuploidies, and defects in recombination are strictly related to meiotic non-disjunction [1]. The possible reason for the increased predisposition to aneuploidy in oocytes when compared with sperm [2] resides in the profound dissimilarity between male and female gametogenesis. While male meiosis is a continuous process, females are born with a complete set of immature oocytes in a quiescent state, and terminate the maturation process in adulthood. In both genders, the irst step of meiosis is DNA replication followed by two rounds of cell division that lead to the formation of haploid gametes. In prophase I, homologous chromosomes align, form chiasmata, and recombination occurs.At the end ofMI, homologs segregate, whereas sister chromatids separate in meiosis II (MII), the second meiotic division. In spermatogenesis, this process initiates after puberty and continues throughout male lifetime, whilst in the female, meiosis begins during fetal development, arrests at prophase I before birth, and resumes prior to ovulation at the luteinizing hormone (LH) surge. For some oocytes, this last phase may never happen, whereas for others it will only occur ater several years, or even decades later. It is postulated that the long time interval of oocyte quiescence, elapsing between meiotic arrest in the fetus and each ovulation cycle, could be responsible for the increased incidence of aneuploidy.

A reduced time interval of oocyte exposure to spermatozoa was investigated to assess whether it could enhance oocyte development and improve embryo viability, especially in cases of male factor infertility. A total of 167 patients were included in a prospective randomized study. They were randomly allocated to two major study groups, A (n = 85) and B (control group; n = 82). The oocytes from group A patients were exposed to spermatozoa for only 1 h; those from group B were exposed for 16 h. The two study groups were then subdivided according to semen quality for further analysis of the results. Significantly higher percentages were obtained in group A than in group B in terms of the fertilization rate (74 versus 68%, P < 0.025), cleavage rate (53 versus 41%, P < 0.005), pregnancy rate (27 versus 12%, P < 0.05) and implantation rate (11 versus 6%, P < 0.05). In addition, an increased fertilization rate was achieved in oocytes exposed to male factor spermatozoa for only 1 h compared with the conventional incubation period (78 versus 65%, P < 0.01). Advanced cellular stages (55 versus 41%, P < 0.02) and higher implantation rates (13 versus 4%, P < 0.05) were attained in the subgroup whose oocytes were exposed to normal spermatozoa for 1 h compared with the male factor spermatozoa with the standard culture interval. The higher fertilization rates, enhanced embryo development and viability achieved in group A indicate that prolonged exposure of oocytes to high concentrations of spermatozoa is detrimental, decreasing

Human oocyte development was evaluated after a reduced time exposure to spermatozoa in vitro. A total of 119 patients were assigned to two study groups in a randomized prospective study in which each patient's oocytes were exposed to spermatozoa for either 1 h (group 1 - 58 patients) or the standard 16 h incubation period (group 2 - 61 patients). The fertilization rate obtained in group 1 was higher than in group 2 (285/393, 73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyte interaction occurs within 1 h. This was confirmed in a study in vitro using fluorescently labelled spermatozoa and normal oocyte-cumulus complexes. Spermatozoa enter the cumulus complex within 15 min, traverse the cumulus layer within 3 h, and first appear in the oocyte cortex at 4 h post-insemination. The incidence of polyspermy was higher in oocytes exposed to spermatozoa for 16 h (3%) than for 1 h (1%). There was no difference in the cleavage rate or morphological characteristics of embryos from both study groups. However, when evaluating the timing of embryo development, group 1 generated a significantly higher percentage of four to five cell embryos when compared to group 2 (55 versus 39%; P < 0.001), documented at 40 h post-insemination. The implantation and pregnancy rates for group 1 were 11 and 28%, while the corresponding rates for group 2 were 8 and 15%. This suggests that a reduced exposure of oocyte to spermatozoa favours embryo viability, possibly due to a decrease in potential damage from sperm metabolic waste products.

BACKGROUND: Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. METHODS: In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1 and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. RESULTS: Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1-15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. CONCLUSIONS: This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.

For a comprehensive picture of the meiotic process and to follow up its products, five chromosomes were tested by fluorescent in-situ hybridization in both polar bodies (PB) and corresponding 145 oocytes. Results were obtained in 143 sets and the prediction of euploidy or aneuploidy based on PB analysis was confirmed by direct analysis in 140 oocytes (98%). Concordance for all chromosomes was found in 132 oocytes, while in the remaining eight, at least one chromosome did not reflect the prediction made by the corresponding PB. When restricting the analysis to the 132 fully concordant oocytes, 215 errors were found in PB: 58% in PB1 and 42% in PB2. Premature separation of chromatids occurred in 89% of aneuploid PB1, whereas only 11% of errors derived from bivalent non-disjunction. In 19% of meiosis-I errors, a complementary error in meiosis II compensated the error originated in the first meiotic division. In conclusion, the testing of PB predicted reliably the oocyte's chromosome condition. Although limited to five chromosomes, the follow up of meiosis by fluorescent in-situ hybridization provided a full description of chromosome allocation during the two divisions characterizing the nuclear maturation of the oocyte. One essential requirement for normal embryo development is the presence of 23 pairs of chromosomes, a condition defined as euploidy. An incorrect number of chromosomes, or aneuploidy, is relatively frequent in human oocytes and is rarely compatible with implantation. The aim of this study was to verify the efficiency of polar body (PB) analysis in predicting the oocyte chromosomal status and to estimate meiotic errors for five chromosomes by fluorescent in-situ hybridization (FISH) in the complete set of female meiosis products, PB1, PB2 and oocyte. In all, 145 oocyte sets were analysed with results obtained in 143; the prediction of euploidy or aneuploidy made by PB testing was confirmed in 140 oocytes (98%). Concordance for all chromosomes was found in 132 oocytes; while in eight at least one chromosome did not reflect the prediction made by the corresponding PB. When restricting the analysis to the 132 fully concordant oocytes, 215 errors were found in the PB: 58% in PB1 and 42% in PB2. In 19% of meiosis-I errors, a complementary error in meiosis II compensated the error originated in the first meiotic division and, accordingly, the corresponding oocytes resulted to be normal for the chromosomes involved in the sequential errors. In conclusion, the testing of PB reliably predicted the oocyte chromosome condition. Although limited to five chromosomes, the follow up of meiosis by FISH provided a full description of chromosome allocation during the two divisions characterizing the nuclear maturation of the oocyte.

Background: Many variations in oocyte and embryo grading make inter-laboratory comparisons extremely difficult. This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. Methods: Background presentations about current practice were given. Results: The expert panel developed a set of consensus points to define the minimumcriteria for oocyte and embryomorphology assessment. Conclusions: It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum data set required for the accurate description of embryo development.

A recent study found a significant correlation between endometriosis and non-obstructive forms of Mullerian anomalies. Other studies described an increased miscarriage rate in patients with endometriosis. This study assessed the effect of endometriosis on pregnancy outcome in a group of patients with endometriosis and septate uterus. Spontaneously achieved pregnancies were taken into consideration. The outcome of 179 infertile women who underwent surgery for septate uterus was analysed in a retrospective study. Stage I or II endometriosis was found by laparoscopy in 36 patients. The pregnancy outcomes, before and after metroplasty, of the group of 36 patients with septum and endometriosis were compared with the pregnancy outcomes of 143 patients with septate uterus with no endometriosis. Before metroplasty the incidence of pregnancy loss was 67% in patients without endometriosis and 75% in patients with endometriosis and the difference was not significant. After metroplasty, no significant differences have been found between the two groups, suggesting that endometriosis could be an occasional finding not influencing pregnancy outcome.

In a population of non-obstructive azoospermia patients, the efficacy of microsurgical testicular sperm extraction (microTESE) and conventional TESE was evaluated in a randomized controlled study on 138 testicles, classified and paired in a 48-square table according to the different classes of the following three variables: patient plasma FSH concentration, orchidometry and testicular histology. Sperm retrieval was positive in 21/22 testicles with hypospermatogenesis (11/11, 10/11; microTESE, TESE respectively), in 12/14 with maturation arrest (6/7, 6/7), in 16/22 with incomplete Sertoli cell-only syndrome (8/11, 8/11), and in 16/80 with complete Sertoli cell-only syndrome (11/40, 5/40). Sperm recovery was positive in 5/24 patients with FSH concentration ≥ 3 × maximum value of normal range (N) (4/12, 1/12), in 17/40 patients with 2N ≤ FSH < 3N (9/20, 8/20), in 30/48 patients with N < FSH < 2N (17/24, 13/24), and in 13/26 patients with FSH = N (6/13, 7/13). Regarding orchidometry, sperm recovery was positive in 11/18 testicles with volume (V) ≥ 12 ml (6/9, 5/9), in 27/56 testicles with 8 ml ≤ V < 12 ml (15/28, 12/28), and in 27/64 testicles with V < 8 ml (15/32, 12/32). FSH value and the surgical procedure were the two variables significantly ( P < 0.05) predicting positive sperm retrieval.

Objective: To report a case of nonobstructive azoospermia in which round spermatids recovered from thawed testicular tissue were used for injection. Design: Case report. Setting: Reproductive Medicine Unit, S.I.S.ME.R. Patient(s): A 33-year-old azoospermic man. Intervention(s): Intracytoplasmic sperm injection with frozen-thawed spermatids. Main Outcome Measure(s): Fertilization, embryo cleavage, pregnancy, and delivery. Result(s): Birth of a healthy, chromosomally normal girl. Conclusion(s): Frozen-thawed testicular round spermatids from a patient with a history of incomplete spermatogenesis can maintain their viability and their capacity to fertilize and to lead to full-term pregnancy.

Objective To verify clinical outcome after injection of spermatozoa that have undergone the acrosome reaction (reacted spermatozoa) vs. those still having an intact acrosome (nonreacted spermatozoa). Design Prospective, randomized study. Setting Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. Patient(s) According to a prospective randomization including 71 couples with severe male factor infertility, intracytoplasmic sperm injection (ICSI) was performed under polarized light that permitted analysis of the pattern of birefringence in the sperm head. Twenty-three patients had their oocytes injected with reacted spermatozoa, 26 patient's oocytes were injected with nonreacted spermatozoa, and in 22 patients both reacted and nonreacted spermatozoa were injected. Intervention(s) Intracytoplasmic sperm injection was performed under polarized light to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa. Main Outcome Measure(s) Rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. Result(s) There was no effect on the fertilizing capacity and embryo development of either type of sperm, whereas the implantation rate was higher in oocytes injected with reacted spermatozoa (39.0%) vs. those injected with nonreacted spermatozoa (8.6%). The implantation rate was 24.4% in the group injected with both reacted and nonreacted spermatozoa. The delivery rate per cycle followed the same trend. Conclusion(s) Spermatozoa that have undergone the acrosome reaction seem to be more prone to supporting the development of viable ICSI embryos.

BACKGROUND: Immature human oocytes matured in vitro, particularly those from gonadotrophin stimulated ovaries, are developmentally incompetent when compared with oocytes matured in vivo. This developmental incompetence has been explained as poor oocyte cytoplasmic maturation without any determination of the likely molecular basis of this observation. METHODS: Replicate whole human genome arrays were generated for immature and mature oocytes (matured in vivo and in vitro, prior to exposure to sperm) recovered from women undertaking gonadotrophin treatment for assisted reproduction. RESULTS: More than 2000 genes were identified as expressed at more than 2-fold higher levels in oocytes matured in vitro than those matured in vivo ( P< 0.05, range 4.98 3 10 22 ‐ 2.22 3 10 24 ) and 162 of these are expressed at 10-fold or greater levels ( P< 0.05, range 4.98 3 10 22 ‐1.38 3 10 23 ). Many of these genes are involved in transcription, the cell cycle and its regulation, transport and cellular protein metabolism. CONCLUSIONS: Global gene expression profiling using microarrays and bioinformatics analysis has provided a molecular basis for differences in the developmental competence of oocytes matured in vitro compared with in vivo. The over-abundance of transcripts identified in immature germinal vesicle stage oocytes recovered from gonadotrophin stimulated cycles and matured in vitro is probably due to dysregulation in either gene transcription or post-transcriptional modification of genes. Either mechanism would result in an incorrect temporal utilization of genes which may culminate in developmental incompetence of any embryos derived from these oocytes.

To find whether chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres of human embryos are nonrandomly localized, whether their aneuploidy affects their localization and if eventual early inactivation of chromosome X with peripheral localization is present.Relative distances from the nucleus center and edge of 1,198 fluorescence in situ hybridization signals in 98 human blastomeres were measured in digital images for comparison with a mathematical model of random distribution in spherical nucleus.Comparison with the mathematical model revealed that localization of chromosomes 13, 16, 21, 22, X and Y in normal and aneuploid blastomeres and that of chromosome 18 in normal blastomeres was not significantly different from random distribution. Similarly, chromosome X in blastomeres with more than one X did not appear to have a preferential localization. Only chromosome 18 in aneuploid blastomeres was differently distributed (p0.0001) with a shift to the nuclear periphery (p =or0.0001).Peripheral localization of chromosome 18 in aneuploid blastomeres is related to embryo aneuploidy. Conversely, a peripheral localization of the inactive X chromosome was not found in blastomeres from 3-4 day old embryos. These results open the possibility to improve embryo selection after pre-implantation diagnosis.

To evaluate the influence of inner myometrium fibroids (myomas) on the outcome of IVF cycles, a retrospective agematched controlled study was performed at SISMeR Reproductive Medicine Unit. The study group included 129 IVF/intracytoplasmic sperm injection cycles in 75 patients with one or more intramural and/or submucosal fibroids, while the control group consisted of 129 cycles in 127 patients without fibroids. The two groups were similar for mean oestradiol concentration at human chorionic gonadotrophin administration (1205.16 +/- 874 versus 1395 +/- 821 pg/ml), mean number of transferred embryos (2.02 +/- 0.4 versus 2.14 +/- 0.6) and clinical pregnancy rate (34.9 versus 41.1%). Conversely, the implantation rate was significantly lower in the study group (18.0%) than in the control group (26.5%; chi(2) = 4.81, P < 0.05), whereas the rate of spontaneous abortion demonstrated an opposite trend (40 versus 18.9%; chi(2) = 4.34, P < 0.05). Further research should be aimed at classifying fibroids on the basis of their location, especially when they are positioned in the junctional zone of the myometrium. Whether this classification will be superior in predicting the impact of fibroids on the reproductive outcome should be elaborated in a large multicentric study.

Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca2+ channels in human granulosa cells. One is long-lasting, activates at approximately -20 mV, reaches the peak at approximately +20 mV, has an inactivation time constant of 132.5 +/- 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately -40 mV, peaks at approximately -10 mV, has an inactivation time constant of 38.8 +/- 1.8 msec at -10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 microm Ni2+, but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through L- and T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 microm) reduces the amplitude of the currents through both L-type (-34.7 +/- 6.4%; n = 10) and T-type (-52.6 +/- 7.4%; n = 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.

BACKGROUND: The biopsy of both polar bodies and a blastomere from the same embryo was investigated as an approach aimed at increasing the quantity of DNA available for genetic analysis in preimplantation embryos. METHODS: In 113 cycles, preimplantation genetic diagnosis (PGD) was performed for aneuploidy: 19 cycles underwent polar body biopsy, 32 cycles had both polar body and blastomere biopsy done, and the remaining 62 cycles underwent blastomere biopsy. The chromosomal analysis was performed in a two-round fluorescence in situ hybridization (FISH) protocol with probes specific for the chromosomes X, Y, 13, 15, 16, 18, 21 and 22. RESULTS: The morphological evaluation of the analysed embryos demonstrated similar rates of development irrespective of the biopsy procedure. Accordingly, the implantation rate did not differ significantly in the three biopsy groups and was 15% after polar body biopsy, 26% after the combined biopsy procedures of polar bodies and blastomeres, and 25% after blastomere biopsy. CONCLUSIONS: The removal of a blastomere subsequent to polar body biopsy does not seem to have negative effects on embryo viability. This approach could be especially valuable for a combined diagnosis of aneuploidy and single-gene disorders in preimplantation embryos generated by couples at high reproductive risk.

Objective To verify whether a correlation exists between pronuclear zygote morphology and the chromosomal condition of preimplantation embryos. Design Prospective analysis of pronuclear zygote morphology and preimplantation genetic diagnosis (PGD) for aneuploidy of the resulting embryos. Setting Reproductive medicine unit, day surgery clinic. Patient(s) Seventy-seven patients undergoing 107 PGD cycles because of advanced maternal age (77 cycles) or previous IVF failures (30 cycles). Intervention(s) Evaluation of pronuclear zygote morphology and chromosomal condition of the resulting embryos. Main outcome measure(s) Rate of embryo development, proportion of euploid embryos, and distribution of chromosomal abnormalities. Result(s) The position of pronuclei within the ooplasm, the size and distribution of nucleoli, and the orientation of polar bodies with respect to pronuclei were highly predictive for the presence of complex chromosomal abnormalities in the developing embryos; zygotes with juxtaposed pronuclei, large-size nucleoli, and polar bodies with small angles subtended by pronuclei and polar bodies were the configurations associated with the highest rates of euploidy. Conclusion(s) The combination of the patterns related to pronuclear zygote morphology indicated four configurations where the proportion of chromosomally normal embryos was significantly higher compared with the other configurations, suggesting the validity of this scoring system for the selection of embryos generated by PGD patients.

Education has always been a priority for the European Society of Human Reproduction and Embryology (ESHRE). Many efforts have been dedicated to promoting knowledge of techniques, procedures and strategies in order to ensure use of the highest quality practices in reproductive medicine. The need to develop a set of guidelines was a logical consequence that found its first expression in 1990, when Focus on Reproduction (vol. 1, pp. 10-38) published the first guidelines which were distributed among the membership. Five years later a new, more complete edition with several novel techniques and developments appeared in Human Reproduction (vol. 10, pp. 1246-1271). Both have proved to be invaluable references. Five more years have now passed. The necessity to produce current guidelines for good IVF laboratory practice has provided the strongest motivation. This originated from the increasing awareness that embryologists have a duty to prevent unintentional incidents that might result from poor practice in the laboratory. Therefore, the Embryology Special Interest Group (SIG) undertook to draw up guidelines aimed at giving support and guidance to the laboratory staff. All the aspects required to provide a safe working system were taken into consideration by members of the SIG and their effort produced this document. We hope that it will assist staff in achieving the best clinical outcome for their patients.