A synopsis of global frontiers in fertility preservation.

Since 2007, the Oncofertility Consortium Annual Conference has brought together a diverse network of individuals from a wide range of backgrounds and professional levels to disseminate emerging basic and clinical research findings in fertility preservation. This network also developed enduring educational materials to accelerate the pace and quality of field-wide scientific communication. Between 2007 and 2019, the Oncofertility Consortium Annual Conference was held as an in-person event in Chicago, IL. The conference attracted approximately 250 attendees each year representing 20 countries around the world. In 2020, however, the COVID-19 pandemic disrupted this paradigm and precluded an in-person meeting. Nevertheless, there remained an undeniable demand for the oncofertility community to convene. To maintain the momentum of the field, the Oncofertility Consortium hosted a day-long virtual meeting on March 5, 2021, with the theme of "Oncofertility Around the Globe" to highlight the diversity of clinical care and translational research that is ongoing around the world in this discipline. This virtual meeting was hosted using the vFairs ® conference platform and allowed over 700 people to participate, many of whom were first-time conference attendees. The agenda featured concurrent sessions from presenters in six continents which provided attendees a complete overview of the field and furthered our mission to create a global community of oncofertility practice. This paper provides a synopsis of talks delivered at this event and highlights the new advances and frontiers in the fields of oncofertility and fertility preservation around the globe from clinical practice and patient-centered efforts to translational research.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Influence of Nodal signalling on pluripotency factor expression, tumour cell proliferation and cisplatin-sensitivity in testicular germ cell tumours.

BACKGROUND: Testicular germ cell tumours (TGCTs) are characterised by an overall high cisplatin-sensitivity which has been linked to their continued expression of pluripotency factors. Recently, the Nodal signalling pathway has been implicated in the regulation of pluripotency factor expression in fetal germ cells, and the pathway could therefore also be involved in regulating expression of pluripotency factors in malignant germ cells, and hence cisplatin-sensitivity in TGCTs. METHODS: We used in vitro culture of the TGCT-derived cell line NTera2, ex vivo tissue culture of primary TGCT specimens and xenografting of NTera2 cells into nude mice in order to investigate the consequences of manipulating Nodal and Activin signalling on pluripotency factor expression, apoptosis, proliferation and cisplatin-sensitivity. RESULTS: The Nodal signalling factors were markedly expressed concomitantly with the pluripotency factor OCT4 in GCNIS cells, seminomas and embryonal carcinomas. Despite this, inhibition of Nodal and Activin signalling either alone or simultaneously did not affect proliferation or apoptosis in malignant germ cells in vitro or ex vivo. Interestingly, inhibition of Nodal signalling in vitro reduced the expression of pluripotency factors and Nodal pathway genes, while stimulation of the pathway increased their expression. However, cisplatin-sensitivity was not affected following pharmacological inhibition of Nodal/Activin signalling or siRNA-mediated knockdown of the obligate co-receptor CRIPTO in NTera2 cells in vitro or in a xenograft model. CONCLUSION: Our findings suggest that the Nodal signalling pathway may be involved in regulating pluripotency factor expression in malignant germ cells, but manipulation of the pathway does not appear to affect cisplatin-sensitivity or tumour cell proliferation.

Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes.

STUDY QUESTION: Does experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation? SUMMARY ANSWER: Inhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries. WHAT IS KNOWN ALREADY: Sex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4. STUDY DESIGN, SIZE, DURATION: FGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 µM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined. PARTICIPANTS/MATERIALS, SETTING, METHODS: Gonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government's support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTION: Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? SUMMARY ANSWER: Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months. WHAT IS KNOWN ALREADY: Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful. STUDY DESIGN, SIZE, DURATION: In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8-1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4-6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids. MAIN RESULTS AND THE ROLE OF CHANCE: Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants. WIDER IMPLICATIONS OF THE FINDINGS: In this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by the ITN Marie Curie Programme 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy.

STUDY QUESTION: Does chemotherapy exposure (with or without alkylating agents) or primary diagnosis affect spermatogonial quantity in human prepubertal testicular tissue? SUMMARY ANSWER: Spermatogonial quantity is significantly reduced in testes of prepubertal boys treated with alkylating agent therapies or with hydroxyurea for sickle cell disease. WHAT IS KNOWN ALREADY: Cryopreservation of spermatogonial stem cells, followed by transplantation into the testis after treatment, is a proposed clinical option for fertility restoration in children. The key clinical consideration behind this approach is a sufficient quantity of healthy cryopreserved spermatogonia. However, since most boys with malignancies start therapy with agents that are not potentially sterilizing, they will have already received some chemotherapy before testicular tissue cryopreservation is considered. STUDY DESIGN, SIZE, DURATION: We examined histological sections of prepubertal testicular tissue to elucidate whether chemotherapy exposure or primary diagnosis affects spermatogonial quantity. Quantity of spermatogonia per transverse tubular cross-section (S/T) was assessed in relation to treatment characteristics and normative reference values in histological sections of paraffin embedded testicular tissue samples collected from 32 consecutive boy patients (aged 6.3 ± 3.8 [mean ± SD] years) between 2014 and 2017, as part of the NORDFERTIL study, and in 14 control samples (from boys aged 5.6 ± 5.0 [mean ± SD] years) from an internal biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prepubertal boys in Sweden, Finland and Iceland who were facing treatments associated with a very high risk of infertility, were offered the experimental procedure of testicular cryopreservation. Exclusion criteria were testicular volumes >10 ml and high bleeding or infection risk. There were 18 patients with a diagnosis of malignancy and 14 patients a non-malignant diagnosis. While 20 patients had the testicular biopsy performed 1-45 days after chemotherapy, 12 patients had not received any chemotherapy. In addition, 14 testicular tissue samples of patients with no reported testicular pathology, obtained from the internal biobank of the Department of Pathology at Karolinska University Hospital, were included as control samples in addition to reference values obtained from a recently published meta-analysis. The quantity of spermatogonia was assessed by both morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents or with hydroxyurea for sickle cell disease. The mean S/T values in boys exposed to alkylating agents (0.2 ± 0.3, n = 6) or in boys with sickle cell disease and exposed to hydroxyurea (0.3 ± 0.6, n = 6) were significantly lower (P = 0.003 and P = 0.008, respectively) than in a group exposed to non-alkylating agents or in biobank control samples (1.7 ± 1.0, n = 8 and 4.1 ± 4.6, n = 14, respectively). The mean S/T values of the testicular tissue samples included in the biobank control group and the patient group exposed to non-alkylating agents were within recently published normative reference values. LIMITATIONS, REASONS FOR CAUTION: Normal testicular tissue samples included in this study were obtained from the internal biobank of Karolinska University Hospital. Samples were considered normal and included in the study if no testicular pathology was reported in the analysed samples. However, detailed information regarding previous medical treatments and testicular volumes of patients included in this biobank were not available. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes, for the first time, spermatogonial quantity in a prepubertal patient cohort just before and after potentially sterilizing treatments. Boys facing cancer and cytotoxic therapies are regarded as the major group who will benefit from novel fertility preservation techniques. There are no previous reports correlating spermatogonial quantity to cumulative exposure to alkylating agents and anthracyclines (non-alkylating agents) and no information about the timing of cytotoxic exposures among this particular patient cohort. For prepubertal boys in whom fertility preservation is indicated, testicular tissue should be obtained before initiation of chemotherapy with alkylating agents, whilst for those with sickle cell disease and treated with hydroxyurea, this approach to fertility preservation may not be feasible. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from The Swedish Childhood Cancer Foundation (PR2016-0124; TJ2016-0093; PR2015-0073, TJ2015-0046) (J.-B.S. and K.J.), the Jane and Dan Olssons Foundation (2016-33) (J.-B.S.), the Finnish Cancer Society (K.J.), the Foundation for Paediatric Research (J.-B.S.), Kronprinsessan Lovisas Förening För Barnasjukvård/ Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation (J.-B.S.), the Väre Foundation for Paediatric Cancer Research (K.J.) and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Ibuprofen is deleterious for the development of first trimester human fetal ovary ex vivo.

STUDY QUESTION: Does ibuprofen use during the first trimester of pregnancy interfere with the development of the human fetal ovary? SUMMARY ANSWER: In human fetuses, ibuprofen exposure is deleterious for ovarian germ cells. WHAT IS KNOWN ALREADY: In utero stages of ovarian development define the future reproductive capacity of a woman. In rodents, analgesics can impair the development of the fetal ovary leading to early onset of fertility failure. Ibuprofen, which is available over-the-counter, has been reported as a frequently consumed medication during pregnancy, especially during the first trimester when the ovarian germ cells undergo crucial steps of proliferation and differentiation. STUDY DESIGN, SIZE, DURATION: Organotypic cultures of human ovaries obtained from 7 to 12 developmental week (DW) fetuses were exposed to ibuprofen at 1-100 µM for 2, 4 or 7 days. For each individual, a control culture (vehicle) was included and compared to its treated counterpart. A total of 185 individual samples were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian explants were analyzed by flow cytometry, immunohistochemistry and quantitative PCR. Endpoints focused on ovarian cell number, cell death, proliferation and germ cell complement. To analyze the possible range of exposure, ibuprofen was measured in the umbilical cord blood from the women exposed or not to ibuprofen prior to termination of pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: Human ovarian explants exposed to 10 and 100 µM ibuprofen showed reduced cell number, less proliferating cells, increased apoptosis and a dramatic loss of germ cell number, regardless of the gestational age of the fetus. Significant effects were observed after 7 days of exposure to 10 µM ibuprofen. At this concentration, apoptosis was observed as early as 2 days of treatment, along with a decrease in M2A-positive germ cell number. These deleterious effects of ibuprofen were not fully rescued after 5 days of drug withdrawal. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed in an experimental setting of human ovaries explants exposed to the drug in culture, which may not fully recapitulate the complexity of in vivo exposure and organ development. Inter-individual variability is also to be taken into account. WIDER IMPLICATIONS OF THE FINDINGS: Whereas ibuprofen is currently only contra-indicated after 24 weeks of pregnancy, our results points to a deleterious effect of this drug on first trimester fetal ovaries ex vivo. These findings deserve to be considered in light of the present recommendations about ibuprofen consumption pregnancy, and reveal the urgent need for further investigations on the cellular and molecular mechanisms that underlie the effect of ibuprofen on fetal ovary development.

Coeliac screening in a Scottish cohort of children with type 1 diabetes mellitus: is DQ typing the way forward?

BACKGROUND: Children with type 1 diabetes mellitus (T1DM) are at increased risk of coeliac disease (CD). Recent guidelines indicate coeliac screening should include HLA typing for CD predisposing (DQ2/DQ8) alleles and those negative for these alleles require no further coeliac screening. METHODS: Children (n=176) with T1DM attending clinics across two Scottish regions were screened for HLA DQ2/DQ8 as part of routine screening. Data collected included the frequency of DQ2/DQ8 genotypes and the additional cost of HLA screening. RESULTS: Overall, DQ2/DQ8 alleles were identified in 94% of patients. The additional cost of HLA typing was £3699.52 (£21.02 per patient). All patients with known CD (11/176) were positive for DQ2/DQ8 and all were diagnosed with CD within 5 years of T1DM diagnosis. CONCLUSIONS: The vast majority of children with T1DM have CD-predisposing HLA genotypes limiting the number of patients that can be excluded from further screening. We conclude that HLA genotyping is not currently indicated for CD screening in this population.

Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development.

STUDY QUESTION: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? SUMMARY ANSWER: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. WHAT IS KNOWN ALREADY: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo. STUDY DESIGN, SIZE, DURATION: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology. LIMITATIONS, REASONS FOR CAUTION: The number of samples included in this study was relatively small due to the limited availability of human fetal tissue. WIDER IMPLICATIONS OF THE FINDINGS: The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.

Do phthalates affect steroidogenesis by the human fetal testis? Exposure of human fetal testis xenografts to di-n-butyl phthalate.

CONTEXT: Phthalates are ubiquitous environmental chemicals. Fetal exposure to certain phthalates [e.g. di-n-butyl phthalate (DBP)] causes masculinization disorders in rats, raising concern for similar effects in humans. We investigated whether DBP exposure impairs steroidogenesis by the human fetal testis. OBJECTIVE: The aim of the study was to determine effects of DBP exposure on testosterone production by normally growing human fetal testis xenografts. DESIGN: Human fetal testes (14-20 wk gestation; n=12) were xenografted into castrate male nude mice that were treated for 4-21 d with vehicle, or 500 mg/kg·d DBP, or monobutyl phthalate (active metabolite of DBP); all mice were treated with human chorionic gonadotropin to mimic normal human pregnancy. Rat fetal testis xenografts were exposed for 4 d to DBP as a positive control. MAIN OUTCOME MEASURES: Testosterone production was assessed by measuring host serum testosterone and seminal vesicle (SV) weights at termination, plus testis gene expression (rats). RESULTS: Human fetal testis xenografts showed similar survival (∼80%) and total graft weight (8.6 vs. 10.1 mg) in vehicle and DBP-exposed hosts, respectively. Serum testosterone (0.56 vs. 0.64 ng/ml; P>0.05) and SV weight (67.2 vs. 81.9 mg; P>0.05) also did not differ. Exposure to monobutyl phthalate gave similar results. In contrast, exposure of rat fetal xenografts to DBP significantly reduced SV weight and testis Cyp11a1/StAR mRNA expression and lowered testosterone levels, confirming that DBP exposure can inhibit steroidogenesis in xenografts, further validating the negative findings on testosterone production in the human. CONCLUSIONS: Exposure of human fetal testes to DBP is unlikely to impair testosterone production as it does in rats. This has important safety and regulatory implications.

Male fertility and strategies for fertility preservation following childhood cancer treatment.

Infertility in the male is a potential complication of childhood cancer treatment for long-term survivors. The risk is dependent primarily on the treatment used, but also on the underlying disease. Chemotherapy (especially alkylating agents) and radiotherapy, even in low doses, may damage the seminiferous epithelium and impair spermatogenesis in both children and adults. Leydig cell function and testosterone production are generally preserved after chemotherapy and low dose radiotherapy, whilst larger doses of radiotherapy may result in hypogonadism. Patients treated with potentially gonadotoxic treatments require regular multidisciplinary follow-up including assessment of puberty and gonadal function. Currently the only option available for fertility preservation in young males treated for cancer is semen cryopreservation. For pre-pubertal patients, techniques for fertility preservation remain theoretical and as yet unproven. These include hormonal manipulation of the gonadal environment before treatment, germ cell transplantation and testis xenografting, which have all shown promise in a variety of animal studies. Refinement of these techniques requires investigations in relevant animal models. In the present chapter we include data which suggest that the common marmoset (Callithrix jacchus) monkey, a New World primate, exhibits important parallels with human testicular development and may help us to understand why the pre-pubertal testis is vulnerable to effects of cytotoxic therapy on future fertility.

Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human.

BACKGROUND: Testicular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans. METHODS: Immunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2gamma, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat. RESULTS: In marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period. CONCLUSIONS: The marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

Public health investigations of Salmonella Enteritidis in catering raw shell eggs, 2002-2004.

AIMS: In response to a dramatic change in the epidemiology of Salmonella Enteritidis in England and Wales thought to be associated with raw shell eggs, the Health Protection Agency initiated public health investigations to establish the incidence of Salmonella contamination and origin of eggs used by catering premises implicated in outbreaks of Salm. Enteritidis. METHODS AND RESULTS: Between October 2002 and November 2004, 16 971 eggs were sampled and Salmonella were recovered from 3.4%. Salmonella was isolated from 5.5% and 6.3% of Spanish and eggs of unknown origin, respectively, used in catering premises linked to outbreaks, a level significantly higher than that (1.1%) found in nonLion Quality UK eggs sampled. The small sample of UK Lion Quality eggs tested (reflecting their lack of use in premises visited) did not contain Salmonella. Several phage types of Salm. Enteritidis other than phage type 4 (PT 4) were identified with nonUK eggs. CONCLUSIONS: Eggs from Spain were implicated as a major source of infection. Eggs were contaminated more frequently with Salmonella when shells were dirty and/or cracked, and stored at above 8 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Spanish eggs by the catering sector has been identified as a consistent significant factor in many of the outbreaks caused by Salm. Enteritidis nonPT4 in England and Wales during 2002-2004. Advice to caterers and hospitals that raw shell eggs should not be used in food that will either not be cooked or only lightly cooked should be reinforced.

Salmonella and raw shell eggs: results of a cross-sectional study of contamination rates and egg safety practices in the United Kingdom catering sector in 2003.

This study was prompted by epidemiological investigations of the unusual number of Salmonella Enteritidis outbreaks associated with the use of eggs in catering premises in England and Wales during 2002. The aims of the study, performed between April and May 2003, were to establish the rate of Salmonella contamination in raw shell eggs from catering premises, investigate any correlation between the origin and type of eggs and the presence of particular serotypes or phage types (PTs) of Salmonella, and examine the use of raw shell eggs in catering premises in the United Kingdom. A total of 34,116 eggs (5,686 pooled samples of six eggs) were collected from 2,104 catering premises, most of which were eggs produced in the United Kingdom (88%). Salmonella was isolated from 17 pools (0.3%) of eggs. Of these, 15 were Salmonella Enteritidis, which were further characterized to PTs as follows: PT6 (0.1%), PT4 (0.07%), PT12 (0.04%), PT1 (0.04%), and PT14b (0.02%). Salmonella Livingstone and Salmonella Typhimurium definitive type 7 resistant to ampicillin, streptomycin, sulfonamides, and tetracycline were also isolated. The Salmonella contamination rate of eggs produced in the United Kingdom appears to have decreased significantly since 1995 and 1996. This trend is reflected in the decrease of Salmonella Enteritidis and, in particular, Salmonella Enteritidis PT4. The impact of the United Kingdom Food Standards Agency's advice on the use of eggs, issued in January 2003, is discussed.

Microbiological quality of pre-cut fruit, sprouted seeds, and unpasteurised fruit and vegetable juices from retail and production premises in the UK, and the application of HAACP.

A study of ready-to-eat pre-cut fruit, sprouted seeds, and unpasteurised fruit and vegetable juices from retail and production premises was undertaken in the UK to determine the microbiological quality of these products, and to verify the application of hazard analysis and critical control points (HACCP) procedures by food operators. Almost all (99%; 2,075/2,096) samples were of satisfactory/acceptable microbiological quality. Two (0.1%) samples (melon, beansprouts) were of unacceptable quality due to the presence of Listeria monocytogenes at 102 cfu/g or more while a further 19 (0.9%) were unsatisfactory due to Escherichia coli levels in the range of 102 to 106 cfu/g. Neither Salmonella spp. nor E. coli O157 were detected in samples examined. A hazard analysis system was in place in most (85%) premises visited, and in 80% it was documented. Most managers (83%) had received some form of food hygiene training. Minimally processed produce is exposed to a range of conditions during production and distribution, and this may increase the potential for microbial contamination, highlighting the need of applying good hygiene practices from farm to fork to prevent contamination and/or bacterial growth. Such products should be stored and displayed at or below 8 degrees C.

Listeria monocytogenes and listeriosis: a review of hazard characterisation for use in microbiological risk assessment of foods.

Considerable effort has been put into the application of quantitative microbiological risk assessment for Listeria monocytogenes, and data are available for England and Wales (probably more so than most other countries) on the adverse health effects, together with incidence data on different age and risk groups for human L. monocytogenes infections. This paper reviews aspects of Listeria and human listeriosis, especially from a public health perspective and provide hazard characterisation data, i.e. the qualitative and/or quantitative evaluation of the adverse health effect associated with the hazard, which is the relationship between exposure levels (dose) and frequency of illness. The majority of cases of human listeriosis are food-borne; however, the disease process is complex with multiple routes of infection. The dose-response relationship is poorly understood, and data from human volunteer studies are not available and would be unethical to produce. Data are available from a range of different animal and in vitro models, although these poorly mimic the natural disease process in route of infection, end point, host and history of prior exposure to the bacterium. Epidemiological data provide some information on infective doses and dose responses, but because of the characteristics of the disease (the hugely variable and potentially very long incubation periods, the low attack rates and the rarity of identification of specific food vehicles), this also provides limited data for calculation of dose responses. There is some, albeit limited, evidence for strain variation, but this is an area of considerable uncertainty despite great advances in the genetic basis of the virulence of this bacterium, and almost all strains seem capable of causing serious disease. A variety of mathematical approaches have been used to model dose responses. The review is written to provide a clinical and epidemiological background to the mathematically oriented, as well as to outline the mathematical approaches to those interested in food-borne infection.

Microbiological examination of ready-to-eat cold sliced meats and pâté from catering and retail premises in the UK.

AIMS: To establish the microbiological quality of cold ready-to-eat sliced meats and pâté from catering and retail premises, and investigate links hypothesized between foodborne Campylobacter infection and the consumption of cold sliced meats. METHODS AND RESULTS: A total of 4078 cold meat and pâté samples were collected and examined according to a standardized protocol. Comparison with published microbiological guidelines revealed that most ready-to-eat meat and pâté samples (75%) were of satisfactory/acceptable microbiological quality and 25% were of unsatisfactory/unacceptable quality. Two cold meat samples (<1%) were of unacceptable microbiological quality because of the presence of Campylobacter jejuni in 25 g and Listeria monocytogenes at 3.4 x 104 CFU g-1. CONCLUSIONS: Acceptable microbiological quality was associated with premises where the management was trained in food hygiene and those that had hazard analysis in place. Poor microbiological quality was associated with storage above 8 degrees C, presliced meats, infrequent cleaning of slicing equipment and poor control of practices that may lead to cross contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides important information about the microbiological quality of cold ready-to-eat meats and pâté. It also assists caterers, retailers, enforcement officers and policy makers to understand how different food safety practices affect microbiological quality.

Microbiological quality of open ready-to-eat salad vegetables: effectiveness of food hygiene training of management.

During September and October 2001, a microbiological study of open, ready-to-eat, prepared salad vegetables from catering or retail premises was undertaken to determine their microbiological quality. The study focused on those salad vegetables that were unwrapped and handled either by staff or customers in the premises where the sample was taken. Examination of salad vegetables from food service areas and customer self-service bars revealed that most (97%; 2,862 of 2,950) were of satisfactory or acceptable microbiological quality, 3% (87) were of unsatisfactory microbiological quality because of Escherichia coli levels in the range of 10(2) to 10(5) colony-forming units per gram. One (<1%) sample was of unacceptable microbiological quality because of the presence of Listeria monocytogenes at 840 colony-forming units per gram. The pathogens E. coli O157, Campylobacter spp., and salmonellas were not detected in any of the samples examined. The display area for most food service and preparation areas (95%) and self-service salad bars (98%) that were visited was judged to be visibly clean by the sampling officer. Most self-service bars (87%) were regularly supervised or inspected by staff during opening hours, and designated serving utensils were used in most salad bars (92%) but in only a minority of food service areas (35%). A hazard analysis system was in place in most (80%) premises, and in 61%, it was documented. Most (90%) managers had received food hygiene training. A direct relationship was shown between increased confidence in the food business management and the presence of food safety procedures and the training of management in food hygiene.

Study of cleaning standards and practices in food premises in the United Kingdom.

A study was undertaken to determine the microbiological status of surfaces used in the preparation of ready-to-eat foods, and to assess cleaning standards and practices in food premises in the UK. A total of 6,533 environmental samples were examined from 1,502 catering (such as restaurants, cafés, and sandwich bars) or retail premises (such as butchers, delicatessens, and bakers): 2,033 samples from chopping/cutting boards, 2,009 from worktop surfaces, 1,359 from food containers, and 1,132 from cleaning cloths. Cleaning cloths were more heavily contaminated with bacteria (Aerobic Colony Count (ACC), Enterobacteriaceae, E. coli, and Staph. aureus) compared to surfaces sampled. Campylobacter spp. were detected in two (0.2%) and Salmonella spp. from one (0.1%) of the cleaning cloths. Surfaces that were visually dirty, wet, last cleaned over 24 hours ago, and boards that were scored or damaged were found to have higher levels of bacteria. A hazard analysis system was in place in most (70%) food premises visited, and in 52% it was documented. Most managers (89%) had received some form of food hygiene training. Documented cleaning schedules and cleaning records were only present in approximately half (55% and 44%, respectively) of the premises. Most did not have separate implements for cleaning raw and ready-to-eat food areas (67%), or stored cleaning equipment for high risk (ready-to-eat food) areas away from those used in low risk (raw, non ready-to-eat food) areas (70%). Deficiencies in the correct use of cleaning products, such as the minimum contact time for disinfectants, were identified. Surface samples (chopping/cutting boards, worktops, and food containers) and cleaning cloths with ACC levels in excess of 10(3) cfu/cm2, swab or ml were associated with premises that did not have management food hygiene training, hazard analysis, cleaning schedules or cleaning records in place, and with little or no confidence in the food business management of food hygiene as indicated by Local Authority Inspectors' Confidence in Management scores.