Several commercially improved strains of Penicillium chrysogenum have been shown to carry amplifications of the entire penicillin biosynthesis gene cluster. Analysis previously carried out using the strain BW 1890 has here been extended to the characterisation of other members of the SmithKline Beecham strain improvement series. We have determined the length of the amplicon to be 57.4 kb and shown a general increase in copy number and penicillin titre through the series. Sequence analyses of the promoter regions of the acvA, ipnA and aat genes in the high titre strain BW 1901, and comparisons with wild-type sequences have not identified any potentially titre-enhancing mutations. In addition, cDNA screening has failed to identify any further transcribed elements within the co-amplified region. The homogeneity of hybridisation patterns and the identification and analysis of a single copy revertant has shown that the amplification is of a direct tandem nature and we propose a model of chromatid misalignment and recombination as its mode of generation. Hybridisation analysis of penicillin non-producing mutants has indicated the loss, in all those investigated, of the entire penicillin biosynthesis gene cluster, similarities between the deletion junctions in these strains and comparison with previously published data indicating the presence of recombinogenic regions flanking the penicillin biosynthesis gene cluster.

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.

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.

The aim of PGD is to test the preimplantation embryo for specific conditions for couples at risk of transmitting that genetic abnormality to their offspring.The couple need to go through IVF procedures to generate embryos in vitro.The embryos can be biopsied at either the zygote, cleavage or blastocyst stage. PGS uses the same technology to screen for chromosome abnormalities in embryos from patients going through IVF procedures as a method of embryo selection. Chromosome analysis was originally performed using fluorescent in situ hybridisation (FISH) which has now been replaced by array comparative genomic hybridisation or next generation sequencing (NGS) For the diagnosis of single gene defects, the polymerase chain reaction (PCR) has been used and has become highly developed over the years. More recently, SNP arrays and karyomapping have been introduced. PGD/PGS require partnership between IVF laboratories and diagnostic centres. As diagnosis can be performed using a variety of strategies with different technologies, 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 Key wordsQuality control, Preimplantation genetic diagnosis 3 The current status of PGD and PGSInitial clinical application of PGD PGD was first performed in 1989 and since this time, genetic testing has seen major advances. PGD was developed as an alternative to prenatal diagnosis, for couples at risk of transmitting a genetic abnormality to their children.Couples have to go through IVF procedures to generate embryos in vitro, even though many of the couples that go through PGD are fertile. The embryos can be biopsied by the embryologists at the zygote stage (removal of the first and second polar body), cleavage stage (removal of 1-2 blastomeres from the 6-8 cell embryo) and blastocyst stage (removal of some trophectoderm cells) (Harton et al., 2011a). Up until recently, almost all PGD cycles were performed on blastomeres after cleavage stage biopsy (Harper et al, 2012, Moutou et al, 2014, but numerous studies have found that cleavage stage embryos exhibit high levels of chromosomal mosaicism, which means that biopsied cells may not be representative of the rest of the embryo (Harper et al, 1995, Munne et al, 1995, Fragouli et al, 2011, Taylor et al, 2014a. This is especially important when trying to perform PGD for a chromosome abnormality. Polar body biopsy is rarely used as it only gives genetic information on the maternal genome. In recent years, the IVF community have seen an increase in the use of blastocyst transfer (Glujovsky et al, 2012) and this has been reflected in the increased use of blastocyst biopsy for PGD (Moutou et al, 2014).The genetic testing should be performed by a specialised genetic testing laboratory. The very first cases of PGD wer...

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.

In man high levels of aneuploidy are seen in spontaneous abortions. Very few autosomal trisomies survive to birth, the three most common being those for chromosome 13, 18 and 21 giving rise to the syndromes named Patau, Edwards and Down respectively. Since the majority of these spontaneously abort, what makes the survivors different from the aborters? Could it be that they have tissue specific mosaicism with the additional normal cell line supporting survival? In this study fluorescence in situ hybridisation was used as a convenient way to detect trisomy in interphase cells. To study the level of mosaicism across gestation, different tissues from 21 trisomic foetuses were analysed using probes for chromosome 13, 18, 21, X and Y. Two trisomy 18 foetuses exhibited mosaicism. Two others, one trisomy 13 and one trisomy 18 had mosaic placentas. There was no clear association between the limited mosaicism seen and severity of the phenotype. We conclude that at least for this sample set, tissue-specific mosaicism was not likely to be responsible for potential survival to birth.

Study question What is currently being taught in United Kingdom (UK) secondary schools relating to sex and fertility and what are students’ experiences of this education? Summary answer There are large gaps in the UK’s biology curriculum relating to sex and fertility education with important topics being neglected. What is known already Sex and fertility education is essential to enable people to make informed choices about family building. This is especially important as maternal and paternal age is increasing globally. School is an important source of this education but sex and fertility education is often minimal. In order to optimise people’s contraceptive behaviour and fertility planning, an understanding of the reproductive cycle, basic physiology of fertility and preconception health is required. Fertility education interventions have been shown to improve fertility knowledge and decrease planned ages of childbearing among young adults but only if repeated. Study design, size, duration This study aimed to evaluate the current biology curricula relating to sex and fertility education at GCSE (General Certificate of Secondary Education) and A level (Advanced Level) in the UK and to determine 16-17-year-old students’ experiences of their sex and fertility education. This year group was chosen as we are interested in what students have learnt by the end of their mandatory education (years 1 to 11), and their experiences of this education. Participants/materials, setting, methods The analysis of the curricula was conducted using the most recently published specifications for science and biology at GCSE and biology at A level for the Awarding Bodies that dominate the GCSE and A-level market in the UK. The school survey included a 47-item online survey distributed to year 12 students in four secondary schools across England. In total, 244 students participated in the survey. Main results and the role of chance There are six Awarding Bodies in the UK that set the examinations for GCSE and A-level students. At GCSE level, the hormonal control of the menstrual cycle, contraception and ART are taught within the human reproduction section of the biology curriculum. STIs are used as examples of communicable diseases, but pregnancy does not feature other than as a consequence of contraceptive failure. At A level, there is generally less teaching of relationships, sexuality and fertility-related topics than at GCSE. The results of the school survey showed that some topics, notably puberty, the menstrual cycle, contraception and STIs, were more likely to be learnt in school. However, topics such as endometriosis, menopause, miscarriage and polycystic ovarian syndrome were more likely to be learnt outside school. Abortion was the most common topic learnt outside school, followed by puberty. The most popular sources of sex education outside school were the internet and social media. In the students’ responses to how they think sex and fertility education can be improved, six themes became apparent: LGBTQ + (lesbian, gay, bisexual, transgender, queer and others) inclusivity; topic variety; logistical improvements; attitudes towards sex; gender equality; and applicability to real life. Limitations, reasons for caution The COVID-19 pandemic significantly disrupted schools during the 2020/2021 academic year. Consequently, we were unable to distribute the survey to as many schools as planned. We hope to continue this study in the 2021/2022 academic year to allow further comparison between the experiences of different groups of students. Wider implications of the findings Ideally, school sex and fertility education would involve a comprehensive and holistic programme and would provide young people with full, accurate information to prepare them for later life. We hope that the results of our study can be used to improve sex and fertility education for young people. Trial registration number NA

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.Keywords Surplus embryos . Embryo disposal practices . IVF M. Simopoulou and K. Sfakianoudis joint first authorship.