Discarding IVF embryos: reporting on global practices.

PURPOSE: To provide a global scale report on a representative sample of the clinical embryology community depicting the practice of discarding supernumerary IVF embryos. METHODS: A web-based questionnaire titled "Anonymous questionnaire on embryo disposal practices" was designed in order to ensure anonymous participation of practicing clinical embryologists around the world. RESULTS: During a data collection period of 8 months, 703 filled-in questionnaires from 65 countries were acquired. According to the data acquired, the majority of practitioners, dispose of embryos by placing them directly in a trash can strictly dedicated for embryo disposal for both fresh and frozen cycles (39% and 36.7% respectively). Moreover, 66.4% of practitioners discard the embryos separately-case by case-at different time points during the day. Over half of embryologists (54%) wait until day 6 to discard the surplus embryos, while 65.5% do not implement a specially allocated incubator space as a designated waiting area prior to disposal. The majority of 63.1% reported that this is a witnessed procedure. The vast majority of embryologists (93%) do not employ different protocols for different groups of patients. Nonetheless, 17.8% reported the request to perform a ceremony for these embryos. Assessing the embryologists' perspective, 59.5% of participants stated that the embryology practice would benefit from a universally accepted and practiced protocol. CONCLUSION(S): This study uniquely provides insight into global embryo disposal practices and trends. Results highlight the divergence between reported practices, while indicating the significance on standardization of practice, with embryologists acknowledging the need for a universally accepted protocol implementation.

Cairo consensus on the IVF laboratory environment and air quality: report of an expert meeting.

This proceedings report presents the outcomes from an international Expert Meeting to establish a consensus on the recommended technical and operational requirements for air quality within modern assisted reproduction technology (ART) laboratories. Topics considered included design and construction of the facility, as well as its heating, ventilation and air conditioning system; control of particulates, micro-organisms (bacteria, fungi and viruses) and volatile organic compounds (VOCs) within critical areas; safe cleaning practices; operational practices to optimize air quality while minimizing physicochemical risks to gametes and embryos (temperature control versus air flow); and appropriate infection-control practices that minimize exposure to VOC. More than 50 consensus points were established under the general headings of assessing site suitability, basic design criteria for new construction, and laboratory commissioning and ongoing VOC management. These consensus points should be considered as aspirational benchmarks for existing ART laboratories, and as guidelines for the construction of new ART laboratories.

Quality control standards in PGD and PGS.

Preimplantation genetic diagnosis (PGD) aims to test the embryo for specific conditions before implantation in couples at risk of transmitting genetic abnormality to their offspring. The couple must undergo IVF procedures to generate embryos in vitro. The embryos can be biopsied at either the zygote, cleavage or blastocyst stage. Preimplantation genetic screening uses the same technology to screen for chromosome abnormalities in embryos from patients undergoing IVF procedures as a method of embryo selection. Fluorescence in-situ hybridization was originally used for chromosome analysis, but has now been replaced by array comparative genomic hybridization or next generation sequencing. For the diagnosis of single gene defects, polymerase chain reaction is used and has become highly developed; however, single nucleotide polymorphism arrays for karyomapping have recently been introduced. A partnership between IVF laboratories and diagnostic centres is required to carry out PGD and preimplantation genetic screening. Accreditation of PGD diagnostic laboratories is important. Accreditation gives IVF centres an assurance that the diagnostic tests conform to specified standards. ISO 15189 is an international laboratory standard specific for medical laboratories. A requirement for accreditation is to participate in external quality assessment schemes.

Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART.

Adult female acne: a new paradigm.

In the adult female, acne is a chronic condition with a substantial negative psychological, social and emotional impact. Based on time of onset, two subtypes of adult female acne are recognized: 'persistent acne' is a continuation of the disease from adolescence, while 'late-onset acne' first presents in adulthood. The morphological characteristics of adult female acne are often distinct from adolescent acne. In adults, inflammatory lesions (particularly papules, pustules and nodules) are generally more prominent on the lower chin, jawline and neck, and comedones are more often closed comedones (micro cysts). Adult acne is mainly mild-to-moderate in severity and may be refractory to treatment. A holistic approach to acne therapy should be taken in adult females, which combines standard treatments with adjunctive therapy and cosmetic use. A number of factors specific to the adult female influence choice of treatment, including the predisposition of older skin to irritation, a possible slow response to treatment, a high likelihood of good adherence, whether of child-bearing age, and the psychosocial impact of the disease. Adherence to therapy should be encouraged through further patient education and a simplified regimen that is tailored to suit the individual patient's needs and lifestyle. This article reviews the specific characteristics of adult female acne, and provides recommendations for acne therapy in this patient group.

Factors influencing the outcome of intrauterine insemination (IUI): age, clinical variables and significant thresholds.

The aim was to investigate the influence of various biological factors upon the outcome of intrauterine insemination (IUI). The total IUI history (856 cycles) of 352 couples was studied. Live-birth showed a strong negative correlation with female age but no correlation with male age. Antimüllerian hormone (AMH) and antral follicle count (AFC) correlated negatively with female age, and follicle stimulating hormone (FSH) correlated positively. Significant thresholds were found for all three variables, and also for total motile count (TMC) in the prepared sperm. Calculating pregnancy losses per positive pregnancy showed a strong correlation with increasing female age. This was highly significant for biochemical losses but not for fetal heart miscarriages. Male age had no effect on rate of pregnancy loss. In conclusion, female age, FSH, AMH and TMC are good predictive factors for live-birth and therefore relate to essential in vivo steps in the reproductive process.

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

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

ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridization-based PGD.

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of new technologies as well as evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. organization of a PGD centre, fluorescence in situ hybridization (FISH)-based testing, amplification-based testing and polar body and embryo biopsy for PGD/preimplantation genetic screening (PGS). Here, we have updated the sections that pertain to FISH-based PGD. PGS has become a highly controversial technique. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Whereas some believe that PGS does not have a place in clinical medicine, others disagree; therefore, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible. Topics covered in this guideline include inclusion/exclusion criteria for FISH-based PGD testing, referrals and genetic counselling, preclinical validation of tests, FISH-based testing methods, spreading of cells for analysis, set-up of local IVF centre and transport PGD centres, quality control/ quality assurance and diagnostic confirmation of untransferred embryos.

ESHRE PGD consortium best practice guidelines for organization of a PGD centre for PGD/preimplantation genetic screening.

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. Subsequent years have seen the introduction of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written/formulated, the Consortium believed it was timely to update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four documents, including one relating to organization of the PGD centre and three relating to the methods used: DNA amplification, fluorescence in situ hybridization and biopsy/embryology. Here, we have updated the sections on organization of the PGD centre. One area that has continued to expand is Transport PGD, in which patients are treated at one IVF centre, whereas their gametes/embryos are tested elsewhere, at an independent PGD centre. Transport PGD/preimplantation genetic screening (PGS) has a unique set of challenges with respect to the nature of the sample and the rapid turn-around time required. PGS is currently controversial. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Current evidence suggests that PGS at cleavage stages is ineffective, but whether PGS at the blastocyst stage or on polar bodies might show improved delivery rates is still unclear. Thus, in this revision, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible.

ESHRE PGD consortium best practice guidelines for amplification-based PGD.

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. Organization of a PGD centre, fluorescence in situ hybridization-based testing, Amplification-based testing and Polar Body and Embryo Biopsy for PGD/preimplantation genetic screening. Here, we have updated the sections that pertain to amplification-based PGD. Topics covered in this guideline include inclusion/exclusion criteria for amplification-based PGD testing, preclinical validation of tests, amplification-based testing methods, tubing of cells for analysis, set-up of local IVF centre and Transport PGD centres, quality control/quality assurance and diagnostic confirmation of untransferred embryos.

Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.

Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt lucrative. Analysis of gametes prior to the initiation of an IVF cycle may improve the quality of embryos transferred. The clinical and scientific community considers it a matter of urgency to translate the basic science behind how a cell prepares for fertilization into routine clinical practice. However, it is equally important, if not more, to allow the science behind such applications to draw level with its practice before its widespread implementation.

Accreditation of the PGD laboratory.

Accreditation according to an internationally recognized standard is increasingly acknowledged as the single most effective route to comprehensive laboratory quality assurance, and many countries are progressively moving towards compulsory accreditation of medical testing laboratories. The ESHRE PGD Consortium and some regulatory bodies recommend that all PGD laboratories should be accredited or working actively towards accreditation, according to the internationally recognized standard ISO 15189, 'Medical laboratories-Particular requirements for quality and competence'. ISO 15189 requires comprehensive quality assurance. Detailed management and technical requirements are defined in the two major chapters. The management requirements address quality management including the quality policy and manual, document control, non-conformities and corrective actions, continual improvement, auditing, management review, contracts, referrals and resolution of complaints. Technical requirements include personnel competence (both technical and medical), equipment, accommodation and environment, and pre-analytical, analytical and post-analytical processes. Emphasis is placed on the particular requirements of patient care: notably sample identification and traceability, test validation and interpretation and reporting of results. Quality indicators must be developed to monitor contributions to patient care and continual improvement. We discuss the implementation of ISO 15189 with a specific emphasis on the PGD laboratory, highlight elements of particular importance or difficulty and provide suggestions of effective and efficient ways to obtain accreditation. The focus is on the European environment although the principles are globally applicable.

ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008.

The 10th report of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium is presented, documenting cycles collected for the calendar year 2007 and follow-up of the pregnancies and babies born until October 2008 which resulted from these cycles. Since the beginning of the data collections there has been a steady increase in the number of cycles, pregnancies and babies reported annually. For data collection X, 57 centres participated, reporting on 5887 cycles to oocyte retrieval (OR), along with details of the follow-up on 1516 pregnancies and 1206 babies born. A total of 729 OR were reported for chromosomal abnormalities, 110 OR for sexing for X-linked diseases, 1203 OR for monogenic diseases, 3753 OR for preimplantation genetic screening and 92 OR for social sexing. Data X is compared with the cumulative data for data collections I-IX.

The causes of misdiagnosis and adverse outcomes in PGD.

The European Society of Human Reproduction and Embryology PGD Consortium has collected data on PGD cycles and deliveries since 1997. From 15,158 cycles, 24 misdiagnoses and adverse outcomes have been reported; 12/2538 cycles after polymerase chain reaction and 12/12,620 cycles after fluorescence in situ hybridization. The causes of misdiagnosis include confusion of embryo and cell number, transfer of the wrong embryo, maternal or paternal contamination, allele dropout, use of incorrect and inappropriate probes or primers, probe or primer failure and chromosomal mosaicism. Unprotected sex has been mentioned as a cause of adverse outcome not related to technical and human errors. The majority of these causes can be prevented by using robust diagnostic methods within laboratories working to appropriate quality standards. However, diagnosis from a single cell remains a technically challenging procedure, and the risk of misdiagnosis cannot be eliminated.

ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007.

The ninth report of the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium is presented documenting cycles collected for the calendar year 2006 and follow-up of the pregnancies and babies born until October 2007, which resulted from these cycles. Since the beginning of the data collections there has been a steady increase in the number of cycles, pregnancies and babies reported annually. For data collection IX, 57 centres have participated, reporting on 5858 cycles to oocyte retrieval (OR), along with details of the follow-up on 1437 pregnancies and 1206 babies born. Eight hundred and twelve ORs were reported for chromosomal abnormalities, 133 ORs for sexing for X-linked diseases, 931 ORs for monogenic diseases, 3900 ORs for preimplantation genetic screening and 82 ORs for social sexing. Data IX are compared with the cumulative data for data collections I-VIII.

Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH.

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.

ESHRE PGD Consortium data collection VIII: cycles from January to December 2005 with pregnancy follow-up to October 2006.

The eighth report of the European Society of Human Reproduction and Embryology PGD Consortium is presented documenting cycles collected for the calendar year 2005 and follow-up of the pregnancies and babies born until October 2006 which resulted from these cycles. For the first time, the delivery rates for each indication are presented and also the pregnancy rates for each centre are reported anonymously. Since the first data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported annually. For data collection VIII, 39 centres have participated, reporting on 3488 cycles to oocyte retrieval (OR), along with details of the follow-up on 845 pregnancies and 670 babies born. Five hundred and twenty OR were reported for chromosomal abnormalities, 108 OR for sexing for X-linked diseases, 500 OR for monogenic diseases, 2275 OR for preimplantation genetic screening and 85 OR for social sexing. Data VIII is compared with the cumulative data for data collections I-VII.

ESHRE PGD consortium data collection VII: cycles from January to December 2004 with pregnancy follow-up to October 2005.

The seventh report of the ESHRE PGD Consortium is presented documenting cycles collected for the calendar year 2004 and follow-up of the pregnancies and babies born subsequent to these cycles up to October 2005. Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported. For data collection VII, 45 centres have participated, reporting on 3358 cycles to oocyte retrieval (OR), 679 pregnancies and 528 babies born. Five hundred and fifty nine OR were reported for chromosomal abnormalities, 113 OR for sexing for X-linked diseases, 520 OR for monogenic diseases, 2087 OR for PGS, and 79 OR for social sexing. Data VII is compared with the cumulative data for data collections I-VI.

Recent developments in genetics and medically assisted reproduction: from research to clinical applications.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Recent developments in genetics and medically-assisted reproduction: from research to clinical applications†‡.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively-parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.