Medically Assisted Reproduction and Risk of Cancer Among Offspring.

Importance: Cancer is a leading cause of death among children worldwide. Treatments used for medically assisted reproduction (MAR) are suspected risk factors because of their potential for epigenetic disturbance and associated congenital malformations. Objective: To assess the risk of cancer, overall and by cancer type, among children born after MAR compared with children conceived naturally. Design, Setting, and Participants: For this cohort study, the French National Mother-Child Register (EPI-MERES) was searched for all live births that occurred in France between January 1, 2010, and December 31, 2021 (and followed up until June 30, 2022). The EPI-MERES was built from comprehensive data of the French National Health Data System. Data analysis was performed from December 1, 2021, to June 30, 2023. Exposure: Use of assisted reproduction technologies (ART), such as fresh embryo transfer (ET) or frozen ET (FET), and artificial insemination (AI). Main Outcomes and Measures: The risk of cancer was compared, overall and by cancer type, among children born after fresh ET, FET, or AI and children conceived naturally, using Cox proportional hazards regression models adjusted for maternal and child characteristics at birth. Results: This study included 8 526 306 children with a mean (SD) age of 6.4 (3.4) years; 51.2% were boys, 96.4% were singletons, 12.1% were small for gestational age at birth, and 3.1% had a congenital malformation. There were 260 236 children (3.1%) born after MAR, including 133 965 (1.6%) after fresh ET, 66 165 (0.8%) after FET, and 60 106 (0.7%) after AI. A total of 9256 case patients with cancer were identified over a median follow-up of 6.7 (IQR, 3.7-9.6) years; 165, 57, and 70 were born after fresh ET, FET, and AI, respectively. The overall risk of cancer did not differ between children conceived naturally and those born after fresh ET (hazard ratio [HR], 1.12 [95% CI, 0.96 to 1.31]), FET (HR, 1.02 [95% CI, 0.78 to 1.32]), or AI (HR, 1.09 [95% CI, 0.86 to 1.38]). However, the risk of acute lymphoblastic leukemia was higher among children born after FET (20 case patients; HR 1.61 [95% CI, 1.04 to 2.50]; risk difference [RD], 23.2 [95% CI, 1.5 to 57.0] per million person-years) compared with children conceived naturally. Moreover, among children born between 2010 and 2015, the risk of leukemia was higher among children born after fresh ET (45 case patients; HR, 1.42 [95% CI, 1.06 to 1.92]; adjusted RD, 19.7 [95% CI, 2.8 to 43.2] per million person-years). Conclusions and Relevance: The findings of this cohort study suggest that children born after FET or fresh ET had an increased risk of leukemia compared with children conceived naturally. This risk, although resulting in a limited number of cases, needs to be monitored in view of the continuous increase in the use of ART.

[Medium and long-term health outcome of children conceived through in vitro fertilization]. / Conséquences de la fécondation in vitro sur la santé des enfants à moyen et long termes.

MEDIUM AND LONG-TERM HEALTH OUTCOME OF CHILDREN CONCEIVED THROUGH IN VITRO FERTILIZATION. Numerous studies have been carried out in children conceived by in vitro fertilization (IVF) focusing on the occurrence of various alterations in their health. It appears that if children can sometimes be affected by health problems, without a particular type predominating, nevertheless their incidence is relatively moderate and not much greater than in naturally conceived children. The alterations observed in children are not necessarily attributable to IVF insofar as infertile couples may be more at risk of transmitting to their children factors responsible for health disturbances. The mechanisms involved in the occurrence of the observed alterations are poorly understood. If disruptions of epigenetic regulations are most often mentioned, research is still needed to clarify them.

CONSÉQUENCES DE LA FÉCONDATION IN VITRO SUR LA SANTÉ DES ENFANTS À MOYEN ET À LONG TERMES. De nombreuses études ont été menées chez les enfants conçus par fécondation in vitro (FIV), s'intéressant à la survenue de différentes altérations de leur santé. Il en ressort que si les enfants peuvent être parfois atteints de troubles de la santé, sans qu'un type particulier prédomine, leur incidence est néanmoins relativement modérée et pas beaucoup plus importante que chez les enfants conçus naturellement. Les altérations observées chez les enfants ne sont pas forcément imputables à la FIV dans la mesure où les couples infertiles peuvent être plus à risque de transmettre à leurs enfants des facteurs responsables de perturbations de santé. Les mécanismes impliqués dans la survenue des altérations observées sont mal connus. Si des perturbations de régulations épigénétiques sont le plus souvent évoquées, des recherches sont encore nécessaires pour les préciser.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

[CRISPR-Cas9, germinal cells and human embryo]. / CRISPR-Cas9, cellules germinales et embryon humain.

The performance of the molecular tool using CRISPR-Cas9, which makes it possible to induce targeted modifications of the DNA, has found numerous applications in research and open promising prospects in human clinic. CRISPR-Cas9 has been widely used to generate transgenic animals after targeted modification of the genome at the zygotic stage. It was also tested on human embryos on an experimental basis. Although there are potential medical indications that may justify a targeted modification of the embryo or germ cell genome, the uncertainties regarding the efficacy and safety of the method do not allow us to consider implementing such germline gene therapy in the short-term. However, it is necessary to weigh the scientific and ethical issues involved in this approach.

'How to count sperm properly': checklist for acceptability of studies based on human semen analysis.

STUDY QUESTION: Can a tool be developed for authors, reviewers and editors of the ESHRE Journals to improve the quality of published studies which rely on semen analysis data? SUMMARY ANSWER: A basic checklist for authors, reviewers and editors has been developed and is presented. WHAT IS KNOWN ALREADY: Laboratory work which includes semen analysis is burdened by a lack of standardization. This has significant negative effects on the quality of scientific and epidemiological studies, potential misclassification of patients and the potential to impair clinical treatments/diagnoses that rely on accurate semen quality information. Robust methods are available to reduce laboratory error in semen analysis, inducing adherence to World Health Organization techniques, participation in an external quality control scheme and appropriate training of laboratory personnel. However, journals have not had appropriate systems to assess if these methods have been used. STUDY DESIGN, SIZE, DURATION: After discussion at a series of Associate Editor Meetings of the ESHRE Journals the authors of the present text were asked to propose a tool for authors, reviewers and editors of the ESHRE Journals to ensure a high quality assessment of submitted manuscripts which rely on semen analysis data, including a detailed verification of the relevance and the quality of the methods used. PARTICIPANTS/MATERIALS, SETTING, METHODS: N/A. MAIN RESULTS AND THE ROLE OF CHANCE: A basic checklist for authors, reviewers and editors is presented. The checklist contains key points which should be considered by authors when designing studies and which provides essential information for when the submitted manuscript is evaluated. For published articles the answers in the checklist are suitable to be available as supplementary data, which will also reduce the space necessary for technical details in the printed article. LIMITATIONS, REASONS FOR CAUTION: Guidelines such as these should not be used uncritically. It is therefore important that submitting authors, in situations where their study does not comply with the basic requirements for semen analysis, not only explain all methodological deviations but also declare the level of uncertainty in their analyses and how it complies with, or might confound, the aims of the study. WIDER IMPLICATIONS OF THE FINDINGS: The fundamental importance of appropriate and robust methodology to facilitate advances in scientific understanding and patient management and treatment, is now accepted as being paramount. Use of the semen analysis checklist should be part of this process, and when completed and signed by the corresponding author at the time of submitting a manuscript should result in greater transparency, and ultimately uniformity. It is hoped that this initiative will pave the way for wider adoption of the methodology/reporting by other biomedical, epidemiological and scientific journals, and ultimately become the standard of practice for papers reporting semen analysis results obtained in laboratory and clinical andrology. Systems to assist referees, authors and editors to present high quality findings should have a significant impact on the field of reproductive medicine. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained for this work. The authors have no competing interests in relation to the present publication and checklist. TRIAL REGISTRATION NUMBER: N/A.

Alcohol and male reproductive health: a cross-sectional study of 8344 healthy men from Europe and the USA.

STUDY QUESTION: Is there an association between alcohol intake and semen quality and serum reproductive hormones among healthy men from the USA and Europe? SUMMARY ANSWER: Moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels. WHAT IS KNOWN ALREADY: High alcohol intake has been associated with a wide range of diseases. However, few studies have examined the correlation between alcohol and reproductive function and most have been conducted in selected populations of infertile men or have a small sample size and the results have been contradictory. STUDY DESIGN, SIZE, DURATION: A coordinated international cross-sectional study among 8344 healthy men. A total of 1872 fertile men aged 18-45 years (with pregnant partners) from four European cities and four US states, and 6472 young men (most with unknown fertility) aged 18-28 years from the general population in six European countries were recruited. PARTICIPANTS/MATERIALS, SETTING, METHODS: The men were recruited using standardized protocols. A semen analysis was performed and men completed a questionnaire on health and lifestyle, including their intake of beer, wine and liquor during the week prior to their visit. Semen quality (semen volume, sperm concentration, percentage motile and morphologically normal sperm) and serum reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, and inhibin B and free testosterone) were examined. MAIN RESULTS AND THE ROLE OF CHANCE: The participation rate for our populations was 20-30%. We found no consistent association between any semen variable and alcohol consumption, which was low/moderate in this group (median weekly intake 8 units), either for total consumption or consumption by type of alcohol. However, we found a linear association between total alcohol consumption and total or free testosterone in both groups of men. Young and fertile men who consumed >20 units of alcohol per week had, respectively, 24.6 pmol/l (95% confidence interval 16.3-32.9) and 19.7 pmol/l (7.1-32.2) higher free testosterone than men with a weekly intake between 1 and 10 units. Alcohol intake was not significantly associated with serum inhibin B, FSH or LH levels in either group of men. The study is the largest of its kind and has sufficient power to detect changes in semen quality and reproductive hormones. LIMITATIONS, REASONS FOR CAUTION: The participation rate was low, but higher than in most previous semen quality studies. In addition, the study was cross-sectional and the men were asked to recall their alcohol intake in the previous week, which was used as a marker of intake up to 3 months before. If consumption in that week differed from the typical weekly intake and the intake 3 months earlier, misclassification of exposure may have occurred. However, the men were unaware of their semen quality when they responded to the questions about alcohol intake. Furthermore, we cannot exclude that our findings are due to unmeasured confounders, including diet, exercise, stress, occupation and risk-taking behavior. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels which may be due to a changed metabolism of testosterone in the liver. Healthy men may therefore be advised that occasional moderate alcohol intake may not harm their reproductive health; we cannot address the risk of high alcohol consumption of longer duration or binge drinking on semen quality and male reproductive hormones. STUDY FUNDING/COMPETING INTERESTS: All funding sources were non-profitable and sponsors of this study played no role in the study design, in data collection, analysis, or interpretation, or in the writing of the article. The authors have no conflicts of interest.

[Extension of assisted reproductive technologies with donor sperm (ARTD) to non-medical indications]. / Ouverture de l'assistance médicale à la procréation avec sperme de donneur (AMPD) à des indications non médicales.

In France as in other countries, more and more single women and lesbian couples wish to become mothers. To carry through their parenting project they may consult a physician in France and often go abroad in order to get Assisted Reproductive Technologies with donor sperm (ARTD). Should ARTD be available to those women in France? The physician has not to take the decision. In such situations ARTD has no medical indication or contraindication. This assisted procreation raises many questions on children development and well-being. The results of studies made in other countries are often reassuring but their methodologies do not allow any conclusion to be drawn and grey areas persist. Therefore it should be necessary to develop a research effort in the field as it recently started in France. Would ARTD access to women without a male partner be legalized, the law should respect the ethical principles of non-payment and anonymity associated with donation of all body components. In any case, it should also allow an efficient medical care to be performed to ensure under the best conditions the well-being of the children and their mothers.

[Issues surrounding the preservation and subsequent use of transsexual persons' gametes]. / Autoconservation des gamètes de personnes trans- sexuelles et projet parental éventuel.

Some transsexual persons wish to have their gametes frozen before gender transition, in order to preserve their fertility. This measure should be carried out, in strict compliance with the law, in case of orchidectomy, oophorectomy or hysterectomy However, as hormonal treatments do not irreversibly alter gonadal function, the reproductive capacity of trans-sexual persons can be maintained by avoiding surgical sterilization. There is therefore no obvious medical indication for cryopreserving gametes or germinal tissue in the absence of surgical sterilization. Moreover, the use of such cryopreserved gametes would, in principle, be considered mainly by a same-sex couple, something that French law currently prohibits. Regardless of these legal aspects, the issues surrounding the use of cryopreserved gametes, and its consequences, must not be ignored. If transsexual persons who are already parents may find ways of managing the change in both their personal and parental identity, the use of gametes stored prior to gender transition raises issues of identity whose consequences are difficult to assess, especially for the future child. Cryopreservation of gametes or germinal tissue cannot be undertaken without first considering whether their potential use is in keeping with what is, at present, medically and legally possible. In any case, it is up the physician to decide, on a case by case basis, whether or not to implement cryopreservation, taking into account the situation of the persons who request the procedure and their plans for parenthood.

Epigenetic disorders and male subfertility.

OBJECTIVE: To provide a link between epigenetics and male subfertility at the DNA, histone-protamine, and RNA levels and its consequences on fertilization and embryo development. DESIGN: Review of the relevant literature. SETTING: University-based clinical and research laboratories. PATIENT(S): Fertile and infertile men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Critical review of the literature. RESULT(S): Epigenetic markers can be modified in infertile patients. Epigenetic modifications include methylation loss or gain on the global level and on imprinted genes, high levels of histone retention in spermatozoa, and deficiencies in some transcripts involved in spermatogenesis. Interestingly, these abnormalities are all linked together, because DNA methylation maintenance depends on DNA histone-protamine configuration which itself is stabilized by spermatozoal RNAs. CONCLUSION(S): The paternal genome has long been considered to be silent and passive in embryo formation. The epigenetic processes associated with the paternal DNA genome highlights its importance in male fertility as well as for embryo development.

Is the nuclear status of an embryo an independent factor to predict its ability to develop to term?

OBJECTIVE: To determine the prognostic impact of the embryo nuclear status at day 2 among other major morphologic parameters (first cleavage at day 1, number of blastomeres and anuclear fragmentation at day 2) on the birth rate. DESIGN: Retrospective study. SETTING: Hospital IVF department. PATIENT(S): Women undergoing 1,629 day 2 transfers of 2,732 embryos from May 2006 to November 2008. INTERVENTION(S): Four groups according to the embryo nuclear status. MAIN OUTCOME MEASURE(S): Implantation, miscarriage, and birth rates. RESULT(S): Univariate analysis indicated significantly higher birth rates when all blastomeres were mononucleated (15.0%) compared with embryos with not all blastomeres mononucleated (9.2%), embryos without any visible nucleus (7.7%), and embryos where at least one blastomere was multinucleated (4.2%). Multivariate analysis found significant decreased birth rates when multinucleated blastomeres were observed. CONCLUSION(S): Blastomere nuclear status should be added to the kinetic and morphologic criteria traditionally used at day 2 to assess human embryo quality. The presence of multinucleated blastomeres has a negative impact on birth potential. The results argue for integrating the blastomere nuclear status at day 2 with the kinetic and morphologic criteria traditionally used to define the best embryo to transfer. Embryos with a single visible nucleus in all blastomeres should be given priority for transfer when available.

Optimal timing for oocyte denudation and intracytoplasmic sperm injection.

Objectives. To analyze the impact of oocyte denudation and microinjection timings on intracytoplasmic sperm injection (ICSI) outcomes. Study Design. We included ICSI cycles with the following parameters: rank 1 or 2, female age <36 years, male factor infertility, long protocol using GnRH agonist and rFSH for ovarian stimulation, and use of freshly ejaculated sperm (n = 110). Several ICSI parameters were analyzed according to the time between oocyte retrieval and denudation (T(1)) and the time between denudation and ICSI (T(2)) using a statistical logistic regression analysis. Results. Neither T(1) nor T(2) had a significant influence on the Metaphase II (MII) rate but the fertilisation rate (FR) showed a significant improvement when T(1) was longer (optimal results at T(1) = 3 hours) while FR significantly decreased with the increase of T(2). Optimal implantation (IR) and pregnancy (PR) rates were obtained when T(1) was around 2 hours. Conclusion. Incubation of oocytes around 2 hours between retrieval and denudation may not increase MII rate but appears to lead to the optimal combination of FR and IR.

[Contribution of animal models to the study of reproduction, assisted reproductive technologies and of development]. / Apport de l'experimentation animale dans l'étude de la reproduction, de la procréation médicale assistée et du développement.

Children conceived through assisted reproductive technologies (ART) now account for a noteworthy proportion (-2.4%) of births in France. Considerable attention is being paid to the outcome of ART pregnancies. The vast majority of these children are apparently normal. However, they are at an increased risk of minor birth defects, low birth weight, and rare imprinting disorders such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS) and Silver Russel syndrome (SRS). Animal models are important for investigating the possible role of each step of ART (ovarian stimulation, gamete manipulation, in vitro fertilization, embryo culture and embryo transfer) in epigenetic reprogramming This review discusses these issues in the context of epigenetic and developmental abnormalities observed in animals following ART More research is needed on ART-induced errors, focusing not only on genomic imprinting but also on non-imprinted loci, which may help explain some of the more subtle longer-term health effects emerging from studies with animal models.

Serum inhibin-b in fertile men is strongly correlated with low but not high sperm counts: a coordinated study of 1,797 European and US men.

OBJECTIVE: To describe associations between serum inhibin-b and sperm counts, adjusted for effect of time of blood sampling, in larger cohorts than have been previously reported. DESIGN: Cross-sectional studies of spermatogenesis markers. SETTING: Four European and four US centers. PATIENT(S): Fertile men (1,797) were included and examined from October 1996-February 2005. INTERVENTION(S): The study was observational and therefore without any intervention. MAIN OUTCOME MEASURE(S): Associations between inhibin-b and semen variables controlled for time of blood sampling and other covariates. RESULT(S): Inhibin-b decreased about 2.00% per hour from 8 am-12 pm and then about 3.25% per hour from 12 pm-4 pm. There was a strong positive association between inhibin-b levels less than 150 pg/mL and both sperm concentration and total sperm count (slopes of the regression lines were ß=0.011 and ß=0.013 for natural logarithm-transformed sperm concentration and total sperm count, respectively). For inhibin-b levels of 150-300 pg/mL the associations were not as steep (ß=0.002), but still significant. For inhibin-b levels more than 300 pg/mL there was little association to the sperm counts. Neither sperm motility nor morphology was significantly related to inhibin-b level in any group. CONCLUSION(S): Serum inhibin-b levels decrease nonlinearly during the daytime, and are positively correlated with sperm counts, but the predictive power is best when inhibin-b is low.

Modulation of imprinted gene network in placenta results in normal development of in vitro manipulated mouse embryos.

Genomic imprinting regulates the expression of a group of genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting genes from the recently described imprinted gene network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated network of imprinted genes results in a compensatory process capable of correcting potential dysfunction of placenta.