Isodicentric Y chromosomes and sex disorders as byproducts of homologous recombination that maintains palindromes.

Massive palindromes in the human Y chromosome harbor mirror-image gene pairs essential for spermatogenesis. During evolution, these gene pairs have been maintained by intrapalindrome, arm-to-arm recombination. The mechanism of intrapalindrome recombination and risk of harmful effects are unknown. We report 51 patients with isodicentric Y (idicY) chromosomes formed by homologous crossing over between opposing arms of palindromes on sister chromatids. These ectopic recombination events occur at nearly all Y-linked palindromes. Based on our findings, we propose that intrapalindrome sequence identity is maintained via noncrossover pathways of homologous recombination. DNA double-strand breaks that initiate these pathways can be alternatively resolved by crossing over between sister chromatids to form idicY chromosomes, with clinical consequences ranging from spermatogenic failure to sex reversal and Turner syndrome. Our observations imply that crossover and noncrossover pathways are active in nearly all Y-linked palindromes, exposing an Achilles' heel in the mechanism that preserves palindrome-borne genes.

Influence of nitrous oxide added to general anaesthesia on postoperative mortality and morbidity: a systematic review and meta-analysis.

BACKGROUND: Nitrous oxide (N2O) is a common adjuvant to general anaesthesia. It is also a potent greenhouse gas and causes ozone depletion. We sought to quantify the influence of N2O as an adjuvant to general anaesthesia on postoperative patient outcomes. METHODS: We searched Medline, EMBASE, and Cochrane Central for works published from inception to July 6, 2023. RCTs comparing general anaesthesia with or without N2O were included. Risk ratios (RRs) and standardised mean differences (SMDs) were calculated, along with 95% confidence intervals (CIs), using a random-effects model. Outcomes were derived from the Standardised Endpoints for Perioperative Medicine (StEP) outcome set. Primary outcomes were mortality and organ-related morbidity, and secondary outcomes were anaesthetic and surgical morbidity. RESULTS: Of 3305 records, 179 full-text articles were assessed, and 71 RCTs, totalling 22 147 patients, were included in the meta-analysis. Addition of N2O to general anaesthesia did not influence postoperative mortality or most morbidity outcomes. N2O increased the incidence of atelectasis (RR 1.62, 95% CI 1.24 to 2.12) and postoperative nausea and vomiting (RR 1.27, 95% CI 1.15 to 1.40), and decreased intraoperative opioid consumption (SMD -0.19, 95% CI -0.35 to -0.04) and time to extubation (MD -2.17 min, 95% CI -3.32 to -1.03 min). CONCLUSIONS: N2O did not influence postoperative mortality or most morbidity outcomes. Considering the environmental effects of N2O, these findings confirm that current policy recommendations to limit its use do not affect patient safety. SYSTEMATIC REVIEW PROTOCOL: PROSPERO CRD42023443287.

Randomized Controlled Trials in ICU in the Four Highest-Impact General Medicine Journals.

OBJECTIVE: To study ICU trials published in the four highest-impact general medicine journals by comparing them with concurrently published non-ICU trials in the same journals. DATA SOURCES: PubMed was searched for randomized controlled trials (RCTs) published between January 2014 and October 2021 in the New England Journal of Medicine , The Lancet , the Journal of the American Medical Association , and the British Medical Journal. STUDY SELECTION: Original RCT publications investigating any type of intervention in any patient population. DATA EXTRACTION: ICU RCTs were defined as RCTs exclusively including patients admitted to the ICU. Year and journal of publication, sample size, study design, funding source, study outcome, type of intervention, Fragility Index (FI), and Fragility Quotient were collected. DATA SYNTHESIS: A total of 2,770 publications were screened. Of 2,431 original RCTs, 132 (5.4%) were ICU RCTs, gradually rising from 4% in 2014 to 7.5% in 2021. ICU RCTs and non-ICU RCTs included a comparable number of patients (634 vs 584, p = 0.528). Notable differences for ICU RCTs were the low occurrence of commercial funding (5% vs 36%, p < 0.001), the low number of RCTs that reached statistical significance (29% vs 65%, p < 0.001), and the low FI when they did reach significance (3 vs 12, p = 0.008). CONCLUSIONS: In the last 8 years, RCTs in ICU medicine made up a meaningful, and growing, portion of RCTs published in high-impact general medicine journals. In comparison with concurrently published RCTs in non-ICU disciplines, statistical significance was rare and often hinged on the outcome events of just a few patients. Increased attention should be paid to realistic expectations of treatment effects when designing ICU RCTs to detect differences in treatment effects that are reliable and clinically relevant.

Childhood cancer and hematological disorders negatively affect spermatogonial quantity at diagnosis: a retrospective study of a male fertility preservation cohort.

STUDY QUESTION: What is the impact of cancer or hematological disorders on germ cells in pediatric male patients? SUMMARY ANSWER: Spermatogonial quantity is reduced in testes of prepubertal boys diagnosed with cancer or severe hematological disorder compared to healthy controls and this reduction is disease and age dependent: patients with central nervous system cancer (CNS tumors) and hematological disorders, as well as boys <7 years are the most affected. WHAT IS KNOWN ALREADY: Fertility preservation in pediatric male patients is considered based on the gonadotoxicity of selected treatments. Although treatment effects on germ cells have been extensively investigated, limited data are available on the effect of the disease on the prepubertal male gonad. Of the few studies investigating the effects of cancer or hematologic disorders on testicular function and germ cell quantity in prepuberty, the results are inconsistent. However, recent studies suggested impairments before the initiation of known gonadotoxic therapy. Understanding which diseases and at what age affect the germ cell pool in pediatric patients before treatment is critical to optimize strategies and counseling for fertility preservation. STUDY DESIGN, SIZE, DURATION: This multicenter retrospective cohort study included 101 boys aged <14 years with extra-cerebral cancer (solid tumors), CNS tumors, leukemia/lymphoma (blood cancer), or non-malignant hematological disorders, who were admitted for a fertility preservation programme between 2002 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: In addition to clinical data, we analyzed measurements of testicular volume and performed histological staining on testicular biopsies obtained before treatment, at cryopreservation, to evaluate number of spermatogonia per tubular cross-section, tubular fertility index, and the most advanced germ cell type prior to chemo-/radiotherapy. The controls were data simulations with summary statistics from original studies reporting healthy prepubertal boys' testes characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Prepubertal patients with childhood cancer or hematological disorders were more likely to have significantly reduced spermatogonial quantity compared to healthy controls (48.5% versus 31.0% prevalence, respectively). The prevalence of patients with reduced spermatogonial quantity was highest in the CNS tumor (56.7%) and the hematological disorder (55.6%) groups, including patients with hydroxyurea pre-treated sickle cell disease (58.3%) and patients not exposed to hydroxyurea (50%). Disease also adversely impacted spermatogonial distribution and differentiation. Irrespective of disease, we observed the highest spermatogonial quantity reduction in patients <7 years of age. LIMITATIONS, REASONS FOR CAUTION: For ethical reasons, we could not collect spermatogonial quantity data in healthy prepubertal boys as controls and thus deployed statistical simulation on data from literature. Also, our results should be interpreted considering low patient numbers per (sub)group. WIDER IMPLICATIONS OF THE FINDINGS: Cancers, especially CNS tumors, and severe hematological disorders can affect spermatogonial quantity in prepubertal boys before treatment. Consequently, these patients may have a higher risk of depleted spermatogonia following therapies, resulting in persistent infertility. Therefore, patient counseling prior to disease treatment and timing of fertility preservation should not only be based on treatment regimes, but also on diagnoses and age. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Marie Curie Initial Training Network (ITN) (EU-FP7-PEOPLE-2013-ITN) funded by European Commision grant no. 603568; ZonMW Translational Adult stem cell research (TAS) grant no. 116003002. No competing interests. TRIAL REGISTRATION NUMBER: N/A.

The risk of hypogonadism after testicular sperm extraction in men with various types of azoospermia: a prospective cohort study.

RESEARCH QUESTION: What is the risk of hypogonadism in men with obstructive azoospermia, non-obstructive azoospermia (NOA) or Klinefelter syndrome after testicular sperm extraction (TESE)? DESIGN: This prospective longitudinal cohort study was carried out between 2007 and 2015. RESULTS: Around 36% of men with Klinefelter syndrome, 4% of men with obstructive azoospermia and 3% of men with NOA needed testosterone replacement therapy (TRT). Klinefelter syndrome was strongly associated with TRT while no association was found between obstructive azoospermia or NOA and TRT. Irrespective of the pre-operative diagnosis, a higher testosterone concentration before TESE was associated with a lower chance of needing TRT. CONCLUSIONS: Men with obstructive azoospermia or NOA have a similar moderate risk of clinical hypogonadism after TESE, while this risk is much larger for men with Klinefelter syndrome. The risk of clinical hypogonadism is lower when testosterone concentrations are high before TESE.

Distinct prophase arrest mechanisms in human male meiosis.

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

Strains matter: Success of murine in vitro spermatogenesis is dependent on genetic background.

The current strategy to preserve fertility of male prepubertal cancer patients consists of cryopreservation of a testicular tissue biopsy containing spermatogonial stem cells (SSCs). While in humans, fertility restoration strategies from prepubertal testicular tissues are still under investigation and have not yet resulted in complete germ cell differentiation, in mice various studies have described production of sperm and offspring through testicular organ culture and transplantation of in vitro propagated SSCs. Organ culture has shown to be successful in generating mature spermatozoa when using testicular fragments from various mouse strains, including CD1 and C57BL/6 J. Conversely, in vitro proliferation of SSCs from C57BL/6 J mice is highly inefficient when compared to other strains such as DBA2 or hybrid mice of C57BL/6 J and DBA2 with 75% C57BL/6 J background (B6D2F2). In this study, we investigated in vitro spermatogenesis by organ culture using testicular tissue from C57BL/6 J and B6D2F2 mice. Whereas spermatogenesis was initiated and completed in C57BL/6 J fragments, it could not be effectively supported in B6D2F2 testicular tissue. While maturation of Sertoli cells and Leydig cells functionality appeared to be identical between the two strains, in B6D2F2 tissue spermatogenesis did not proceed past the spermatocyte step, followed by a rapid decline of the number of all germ cells in the fragments. This suggests that the spermatogenic potential in vitro is dependent on specialized sites in the genome and therefore the organ culture conditions suboptimal for some strains of mice.

Unraveling transcriptome dynamics in human spermatogenesis.

Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure.

High-quality human preimplantation embryos stimulate endometrial stromal cell migration via secretion of microRNA hsa-miR-320a.

STUDY QUESTION: How do high-quality human preimplantation embryos influence the endometrium to promote their own implantation? SUMMARY ANSWER: High-quality human preimplantation embryos secrete a specific microRNA (miRNA), hsa-miR-320a, which promotes migration of human endometrial stromal cells (hESCs). WHAT IS KNOWN ALREADY: We have previously shown that high-quality human preimplantation embryos excrete unknown factors that influence migration of hESCs. STUDY DESIGN, SIZE, DURATION: Embryo excreted miRNAs, specifically those excreted by high-quality embryos, were identified and their effect on hESCs was determined by measuring the migration capacity and gene expression patterns of primary isolated hESCs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryo conditioned medium (ECM) from routine ICSI procedures was used to identify embryo excreted miRNAs. miRNome analyses were performed on ECM from individually cultured embryos with high morphological quality, with low morphological quality or empty control medium. MiRNA mimics and inhibitors were then used to further study the effect of miRNAs of interest on migration and gene expression of hESCs. Migration assays were performed using hESCs that were obtained from endometrial biopsies performed on hysterectomy specimens from women that received surgery for spotting due to a niche in a cesarean section scar. MAIN RESULTS AND THE ROLE OF CHANCE: By using miRNA mimics and inhibitors, we showed that hsa-miR-320a alone can stimulate migration of decidualized hESCs, accurately resembling the response typically triggered only by high-quality embryos. Transcriptome analysis further demonstrated that this effect is very likely mediated via altered expression of genes involved in cell adhesion and cytoskeleton organization. LIMITATIONS, REASONS FOR CAUTION: The effect of hsa-miR-320a on hESCs was measured in vitro. Further studies on the in vivo effect of hsa-miR-320a are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Implantation failure is one of the major success limiting factors in human reproduction. By secreting hsa-miR-320a, high-quality human preimplantation embryos directly influence hESCs, most likely to prime the endometrium at the implantation site for successful implantation. Together, our results indicate that hsa-miR-320a may be a promising target to further increase success rates in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam University Medical Centers and the Amsterdam Reproduction & Development Research Institute. R.P.B., G.H. and S.M. have a patent on the use of hsa-miR-320a in assisted reproduction treatments pending. TRIAL REGISTRATION NUMBER: N/A.

Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media.

STUDY QUESTION: Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium? SUMMARY ANSWER: We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium. WHAT IS KNOWN ALREADY: Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta. STUDY DESIGN, SIZE, DURATION: To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008-2013 in the Netherlands as reference material. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects. MAIN RESULTS AND THE ROLE OF CHANCE: We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium.groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon. LIMITATIONS, REASONS FOR CAUTION: Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues. WIDER IMPLICATIONS OF THE FINDINGS: It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.

Gonadotrophins or clomiphene citrate in couples with unexplained infertility undergoing intrauterine insemination: a cost-effectiveness analysis.

RESEARCH QUESTION: What is the cost-effectiveness of gonadotrophins compared with clomiphene citrate in couples with unexplained subfertility undergoing intrauterine insemination (IUI) with ovarian stimulation under strict cancellation criteria? DESIGN: A cost-effectiveness analysis alongside a randomized controlled trial (RCT). Between July 2013 and March 2016, 738 couples were randomized to gonadotrophins (369) or clomiphene citrate (369) in a multicentre RCT in the Netherlands. The direct medical costs of both strategies were compared. Direct medical costs included costs of medication, cycle monitoring, insemination and, if applicable, pregnancy monitoring. Non-parametric bootstrap resampling was used to investigate the effect of uncertainty in estimates. The cost-effectiveness analysis was performed according to intention-to-treat. The incremental cost-effectiveness ratio (ICER) between gonadotrophins and clomiphene citrate for ongoing pregnancy and live birth was assessed. RESULTS: The mean costs per couple were €1534 for gonadotrophins and €1067 for clomiphene citrate (mean difference of €468; 95% confidence interval [CI] €464-472). As ongoing pregnancy rates were 31% in women allocated to gonadotrophins and 26% in women allocated to clomiphene citrate (relative risk 1.16, 95% CI 0.93-1.47), the ICER was €21,804 (95% CI €11,628-31,980) per additional ongoing pregnancy with gonadotrophins and €17,044 (95% CI €8998-25,090) per additional live birth with gonadotrophins. CONCLUSIONS: Gonadotrophins are more expensive compared with clomiphene citrate in couples with unexplained subfertility undergoing IUI with adherence to strict cancellation criteria, without being significantly more effective.

Premature expression of the decidualization marker prolactin is associated with repeated implantation failure.

Repeated implantation failure (RIF) is a poorly understood reproductive pathology defined by the inability to achieve a clinical pregnancy in at least three consecutive IVF cycles. In this study, we investigated whether the onset of decidualization, marked by prolactin (PRL) expression, is associated with RIF. We performed a retrospective cohort study using endometrial biopsies from women with idiopathic subfertility, that conceived naturally during the same cycle in which the biopsy was taken (group 1; n = 15) conceived naturally within three months after the biopsy was taken (group 2; n = 20), or unsuccessfully underwent six IUI cycles and three IVF cycles with transfer of at least one high-quality embryo (group 3, RIF; n = 20). Our results demonstrated that immunohistochemical PRL-staining was present in 8/15 women from group 1 (53.3%), in 1/20 women from group 2 (5.0%), and in 11/20 women from group 3 (55.0%). Increased proliferation, analyzed by Ki67 expression, was seen in women that were pregnant during the biopsy, compared to all women combined that were not pregnant (p≤.01). In conclusion, our study demonstrates that premature expression of the decidualization marker PRL during the luteal phase is associated with RIF.

ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture.

Autologous spermatogonial stem cell transplantation is an experimental technique aimed at restoring fertility in infertile men. Although effective in animal models, in vitro propagation of human spermatogonia prior to transplantation has proven to be difficult. A major limiting factor is endogenous somatic testicular cell overgrowth during long-term culture. This makes the culture both inefficient and necessitates highly specific cell sorting strategies in order to enrich cultured germ cell fractions prior to transplantation. Here, we employed RNA-Seq to determine cell type composition in sorted integrin alpha-6 (ITGA6+) primary human testicular cells (n = 4 donors) cultured for up to two months, using differential gene expression and cell deconvolution analyses. Our data and analyses reveal that long-term cultured ITGA6+ testicular cells are composed mainly of cells expressing markers of peritubular myoid cells, (progenitor) Leydig cells, fibroblasts and mesenchymal stromal cells and only a limited percentage of spermatogonial cells as compared to their uncultured counterparts. These findings provide valuable insights into the cell type composition of cultured human ITGA6+ testicular cells during in vitro propagation and may serve as a basis for optimizing future cell sorting strategies as well as optimizing the current human testicular cell culture system for clinical use.

Hormonal medication in medically assisted reproduction: a systematic review of assessments from patients.

Several hormonal fertility medications have comparable effectiveness. A literature review was conducted into patients' assessments regarding seven medication characteristics including 'side effects' and 'ease of use'. Medline, CINAHL and PsycINFO were searched for female fertility patients' written assessments of a hormonal medication. The tools used were appraised and common (i.e. ≥10%) unpleasant consequences were distinguished from rare ones. The 35 eligible studies did not rely on valid and reliable tools and did not provide patient assessments regarding all seven medication characteristics for any of the globally used medications. Evidence on medications for oocyte triggering was absent and for induction of pituitary quiescence it was scarce. Regarding medications for ovarian stimulation and luteal support, evidence on general side effects (mostly headache), local side effects (mostly pain), 'interference with home life' and 'impact on psychological wellbeing' was found. Evidence on 'ease of use' and 'required education' was only identified for medication for ovarian stimulation. Evidence on 'interference with work life' and 'compliance worry' was absent. This review calls for randomized controlled trials questioning patients with valid and reliable tools. In the meantime, this review's summary of the best available evidence can be integrated in decision aids facilitating personalized and informed medication choices.

The relative importance of genetic parenthood.

RESEARCH QUESTION: How much do patients with severe infertility and their gynaecologists value genetic parenthood relative to other key treatment characteristics? DESIGN: A discrete choice experiment included the following treatment characteristics: genetic parenthood, pregnancy rate, curing infertility, maternal health, child health and costs. The questionnaire was disseminated between 2015 and 2016 among Dutch and Belgian patients with severe infertility and their gynaecologists. RESULTS: The questionnaire was completed by 173 patients and 111 gynaecologists. When choosing between treatments that varied in safety, effectiveness and costs, the treatment's ability to lead to genetic parenthood did not affect the treatment preference of patients with severe infertility (n = 173). Genetic parenthood affected the treatment preference of gynaecologists (n = 111) less than all other treatment characteristics. Patients indicated that they would switch to a treatment that did not enable genetic parenthood in return for a child health risk reduction of 3.6%, a cost reduction of €3500, an ovarian hyperstimulation risk reduction of 4.6%, a maternal cancer risk reduction of 2.7% or a pregnancy rate increase of 18%. Gynaecologists made similar trade-offs. CONCLUSIONS: While awaiting replication of this study in larger populations, these findings challenge the presumed dominant importance of genetic parenthood. This raises questions about whether donor gametes could be presented as a worthy alternative earlier in treatment trajectories and whether investments in novel treatments enabling genetic parenthood, like in-vitro gametogenesis, are proportional to their future clinical effect.

Orchidopexy for bilateral undescended testes: A multicentre study on its effects on fertility and comparison of two fixation techniques.

To evaluate fertility potential after orchidopexy for bilateral undescended testis and compare two surgical fixation techniques for effect on fertility. Men older than 22 years who had either tunica albuginea orchidopexy (TAO) or "no-touch" technique (NTO) in childhood for bilateral undescended testis (BUDT) were selected. Participants filled out a questionnaire followed by physical examination, had testicular ultrasound, blood sample and semen analysis. Statistical testing was performed using general linear modelling. Sixty-seven out of 166 individuals responded. Forty-nine completed the questionnaire, and nine (18.3%) reported having fathered children. Thirty-six showed up for further examination, 26 had TAO and 10 NTO. Impaired hormonal spermatogenesis regulation (34.6% vs. 20%), higher subfertility rate (46% vs. 20%) and lower means of motile spermatozoa (58.1 × 106 spz vs. 177.9 × 106 spz) were observed in the TAO versus the NTO group; none of these were statistically significant. Four (15.4%) of the TAO and two (20%) of the NTO group have azoospermia. Although the operation technique did not have a significant impact on fertility, unfavourable outcomes were more common after surgery involving the tunica albuginea of the testis. Larger sample sizes are needed to ascertain whether the trends favouring the NTO technique are of any significance.

The ethics of clinical applications of germline genome modification: a systematic review of reasons.

STUDY QUESTION: What are the reasons for or against the future clinical application of germline genome modification (GGM)? SUMMARY ANSWER: A total of 169 reasons were identified, including 90 reasons for and 79 reasons against future clinical application of GGM. WHAT IS KNOWN ALREADY: GGM is still unsafe and insufficiently effective for clinical purposes. However, the progress made using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated system (Cas) has led scientists to expect to overcome the technical hurdles in the foreseeable future. This has invited a debate on the socio-ethical and legal implications and acceptability of clinical applications of GGM. However, an overview of the reasons presented in this debate is missing. STUDY DESIGN, SIZE, DURATION: MEDLINE was systematically searched for articles published between January 2011 and June 2016. Articles covering reasons for or against clinical application of intentional modification of the nuclear DNA of the germline were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two researchers independently extracted the reported reasons from the articles and grouped them into categories through content analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The systematic search yielded 1179 articles and 180 articles were included. Most papers were written by professionals in ethics, (science) journalism and biomedical sciences. Overall, 169 reasons were identified, including 90 reasons for, and 79 reasons against future clinical application of GGM. None of the included articles mentioned more than 60/169 reasons. The reasons could be categorized into: (i) quality of life of affected individuals; (ii) safety; (iii) effectiveness; (iv) existence of a clinical need or alternative; (v) costs; (vi) homo sapiens as a species (i.e. relating to effects on our species); (vii) social justice; (viii) potential for misuse; (ix) special interests exercising influence; (x) parental rights and duties; (xi) comparability to acceptable processes; (xii) rights of the unborn child; and (xiii) human life and dignity. Considerations relating to the implementation processes and regulation were reported. LIMITATIONS, REASONS FOR CAUTION: We cannot ensure completeness as reasons may have been omitted in the reviewed literature and our search was limited to MEDLINE and a 5-year time period. WIDER IMPLICATIONS OF THE FINDINGS: Besides needing (pre)clinical studies on safety and effectiveness, authors call for a sound pre-implementation process. This overview of reasons may assist a thorough evaluation of the responsible introduction of GGM. STUDY FUNDING/COMPETING INTEREST(S): University of Amsterdam, Alliance Grant of the Amsterdam Reproduction and Development Research Institute (I.D.), and Clinical Center, Department of Bioethics, National Institutes of Health Intramural Research Program (S.H.). There are no competing interests.

A practical blueprint to systematically study life-long health consequences of novel medically assisted reproductive treatments.

In medicine, safety and efficacy are the two pillars on which the implementation of novel treatments rest. To protect the patient from unnecessary or unsafe treatments, usually, a stringent path of (pre) clinical testing is followed before a treatment is introduced into routine patient care. However, in reproductive medicine several techniques have been clinically introduced without elaborate preclinical studies. Moreover, novel reproductive techniques may harbor safety risks not only for the patients undergoing treatment, but also for the offspring conceived through these techniques. If preclinical (animal) studies were performed, efficacy and functionality the upper hand. When a new medically assisted reproduction (MAR) treatment was proven effective (i.e. if it resulted in live birth) the treatment was often rapidly implemented in the clinic. For IVF, the first study on the long-term health of IVF children was published a decade after its clinical implementation. In more recent years, prospective follow-up studies have been conducted that provided the opportunity to study the health of large groups of children derived from different reproductive techniques. Although such studies have indicated differences between children conceived through MAR and children conceived naturally, results are often difficult to interpret due to the observational nature of these studies (and the associated risk of confounding factors, e.g. subfertility of the parents), differences in definitions of clinical outcome measures, lack of uniformity in assessment protocols and heterogeneity of the underlying reasons for fertility treatment. With more novel MARs waiting at the horizon, there is a need for a framework on how to assess safety of novel reproductive techniques in a preclinical (animal) setting before they are clinically implemented. In this article, we provide a blueprint for preclinical testing of safety and health of offspring generated by novel MARs using a mouse model involving an array of tests that comprise the entire lifespan. We urge scientists to perform the proposed extensive preclinical tests for novel reproductive techniques with the goal to acquire knowledge on efficacy and the possible health effects of to-be implemented reproductive techniques to safeguard quality of novel MARs.

Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells.

STUDY QUESTION: Is testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice? SUMMARY ANSWER: Cancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls. WHAT IS KNOWN ALREADY: Spermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically. STUDY DESIGN, SIZE, DURATION: This study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Neonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497-517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR. MAIN RESULTS AND THE ROLE OF CHANCE: We found 9% (95% CI: 2-25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14-45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1-1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Although we attempted to mimic the future clinical application of SSCT in humans as close as possible, the mouse model that we used might not reflect all aspects of the future clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: The absence of an increase in cancer incidence and a decrease in survival of mice that received a testicular transplantation of in vitro propagated SSCs is reassuring in light of the future clinical application of SSCT in humans. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by KiKa (Kika86) and ZonMw (TAS 116003002). The authors report no financial or other conflict of interest relevant to the subject of this article.

pH stability of human preimplantation embryo culture media: effects of culture and batches.

RESEARCH QUESTION: How stable is the pH of human preimplantation embryo culture media during IVF culture and is there variation in pH between batches of culture media? DESIGN: To evaluate pH stability, three batches of three culture media were incubated in triplicate without embryos (sham culture) at CO2 levels recommended by the manufacturers (5% or 6%) for 4 days. To evaluate differences in pH between batches, the pH of three batches of five culture media was measured in triplicate during 1 day of sham culture. Linear mixed models were used for the analysis. RESULTS: An increase in pH during 4 days of culture was found in all three culture media, but the observed increased values were within the generally accepted range for clinical practice (pH 7.2-7.4). One medium was pH 7.1 in the first 2 days, but this was within the range provided by the manufacturer for that medium. Three out of five analysed media showed batch variation in pH that exceeded the generally accepted range for clinical practice. CONCLUSIONS: A relevant difference in pH was found between batches of human preimplantation embryo culture media. This suggests that the CO2 level of incubators may need to be adjusted for new batches of culture medium based on measured pH, to anticipate batch variability and safely accommodate limited pH increase over time. This study was unable to identify the cause of the differences in pH between batches, and further investigation on a larger number of batches and other media seems warranted.