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.

'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.

[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.

[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.

[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.

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.

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.

[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.

Seminal expression of NY-ESO-1 and MAGE-A4 as markers for the testicular cancer.

Testicular germ cell tumours (TGCTs) are the most common malignancies in Caucasian young men and their incidence has increased over the past decades. However, a non-invasive test allowing an early diagnosis of TGCT often proves inaccurate. We have previously shown that two Cancer-Testis Antigens (CTA), namely MAGE-A4 and NY-ESO-1, were expressed by TGCT. As exfoliation of carcinoma in situ (CIS) cells or tumour germ cells from testis into seminal fluid can occur, here we studied the expression of the 2 CTA in semen smears of patients with testicular cancer in comparison with healthy men. Using semen smears from healthy controls (n = 65) and patients diagnosed for testicular tumour (n = 57) and immunological staining, we observed expression of MAGE-A4 and NY-ESO-1 proteins in seminal fluid exfoliated cells. We found a highly statistically significant difference in the ratios of stained cells to the total number of round cells between testicular cancer patients and healthy controls. Multivariable analysis, including sperm parameters and immunostaining on sperm smears, shows the improvement. This technique can provide towards testicular cancer diagnosis when it is included in the current testing regime. However, the fact that expression of these markers was not restricted to foetal germ cells led to detection in the semen of a number of healthy subjects. Although the detection of these CTA could be useful to characterize the sub-type of individual TGCTs better, we stress here that the false positive rate precludes the exclusive employment of these CTA for the early detection of testicular neoplasia.

From ultrastructural flagellar sperm defects to the health of babies conceived by ICSI.

The objective of this retrospective study was to describe a population of patients displaying impaired sperm motility due to ultrastructural flagellar defects and to analyse the intracytoplasmic sperm injection (ICSI) results and neonatal outcomes in this population. The fertilization rate, embryo quality, clinical pregnancy rate, implantation rate, birth rate and perinatal health of babies were determined. Patients (n = 20) were divided into seven categories according to ultrastructural flagellar abnormalities. The type of flagellar abnormality significantly affected the fertilization rate (P <0.025). Two types of flagellar abnormalities showed slower early embryo cleavage kinetics (P <0.001) when axonemal central structures and periaxonemmal columns were abnormal or absent. Of 53 ICSI attempts, 14 resulted in clinical pregnancies (26.4% per cycle) after fresh and frozen embryo transfer. Three (21.4%) of these pregnancies ended in miscarriages and, in the remaining, 12 infants were born (7.2% of transferred embryos). The outcomes differed according to the ultrastructural defect. This study demonstrates that a high proportion of patients could father a child (45.0%). However, flagellar abnormalities appear to influence ICSI results and fetal development.

Human sperm quality and lipid content after migration into normal ovulatory human cervical mucus containing low numbers of leukocytes.

The aim of this study was to investigate whether a relationship exists between the presence of low numbers of leukocytes in normal ovulatory cervical mucus and sperm quality and lipid content after migration. The percentages of live, motile and morphologically normal spermatozoa, movement parameters assessed by computer-aided sperm analysis (CASA), and ionophore-induced acrosome reaction measured by flow cytometry were determined before and after migration. High-performance liquid chromatography with ultraviolet detection was used to measure the sperm lipid content, including the various diacyl subspecies. The number of leukocytes found in solubilized mucus samples was counted using a haemocytometric method. Overall, the presence of leukocytes in the cervical mucus samples did not significantly influence sperm motility and morphology, sperm kinematic parameters, or the sperm content in sphingomyelin or cholesterol. In contrast, after migration, the decrease in various sperm diacyls and the level of induced acrosome reaction was significantly less pronounced in mucus samples containing>or=10(4) leukocytes than in mucus samples with no or rare leukocytes whereas the level of induced acrosome reaction was higher. The present data suggest that the low level of leukocytes found in normal ovulatory cervical mucus could influence the process of sperm lipid remodelling/capacitation.

[Evolution of assisted reproductive technologies]. / Evolution des techniques de l'assistance médicale à la procréation (AMP).

When natural conception is impossible and the underlying problem cannot be treated, medical intervention can reproduce the steps necessary for fertilization and early embryo development. The first known medical action in the field of human reproduction took place at the end of the 18th century, in the form of artificial insemination with the husband's semen, thus dissociating sexual intercourse from procreation. A further upheaval occurred at the end of the 19th century, with the use of donor sperm, separating the notions of genetic descent and parenthood. In the second half of the 20th century, medically assisted procreation saw two major technological advances, namely gamete freezing and in vitro fertilization (IVF). The first child conceived with frozen-thawed sperm was born in 1953, and the first IVF baby in 1978. Fertilization by intracytoplasmic sperm injection (ICSI), first developed in 1992, can overcome many causes of male infertility. The convergence of reproductive biology and genetics has now opened up the possibility of screening for chromosome and gene defects in the embryo, prior to implantation. Thus, assisted reproductive technologies (ART) not only serve as a substitute for natural conception but can also avoid the birth of a disabled child While new technologies continue to extend the available options for infertile couples, they also have the potential to help single women and homosexual couples to have children. These practices are currently only accepted in certain countries. Overall, these new medical technologies have contributed to changing our conception of human reproduction, opening up new paradigms of parenthood and raising new challenges for society.

Influence of sickle cell disease and treatment with hydroxyurea on sperm parameters and fertility of human males.

BACKGROUND: Recent progress in the treatment of sickle cell disease, in particular the use of hydroxyurea, has considerably modified the prognosis of this disease. Many more patients now reach reproductive age. The objective of this study was to assess the potential impact of hydroxyurea on the semen of patients. DESIGN AND METHODS: In this retrospective multicenter study, we evaluated the sperm parameters and fertility of 44 patients and analyzed the potential impact of hydroxyurea. RESULTS: We report data from the largest series so far of semen analyses in patients with sickle cell disease: 108 samples were analyzed, of which 76 were collected before treatment. We found that at least one sperm parameter was abnormal in 91% of the patients before treatment, in agreement with published literature. All sperm parameters seemed to be affected in semen samples collected during hydroxyurea treatment, and this impairment occurred in less than 6 months, later reaching a plateau. Furthermore, after hydroxyurea cessation, while global results in 30 patients were not statistically different before and after hydroxyurea treatment, in four individuals follow-up sperm parameters did not seem to recover quickly and the total number of spermatozoa per ejaculate fell below the normal range in about half the cases. CONCLUSIONS: The observed alterations of semen parameters due to sickle cell disease seem to be exacerbated by hydroxyurea treatment. Until prospective studies reveal reassuring findings, we suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment.

In vitro spermicidal activity of peptides from amphibian skin: dermaseptin S4 and derivatives.

Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC(100) values: 25 microg/ml-l mg/ml). The order was K4S4=S4a>S4>K4S4(1-16)a>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.

[Medically assisted procreation in France; current situation and future prospects]. / La procréation médicalisée en France, état des lieux et perspectives.

Infertility treatment has improved greatly with the advent of biological and medical technologies allowing gamete maturation, fertilization and embryo culture before implantation. The ethical, social and medical issues raised by assisted reproductive technologies (ART) led French legislators in 1994 to define the conditions of ART access and realization. These regulatory measures were recently bolstered by the creation of an Agence de la Biomédecine. In 2005, 123,000 ART treatment cycles were performed in France, all techniques included. They resulted in the birth of 19,026 children. The clinical pregnancy rate ranged from 11 to 24.4% per attempt and the multiple pregnancy rate from 11 to 21%, depending on the technique used. Nowadays, the first priority is to improve the quality of the results, which means controlling the risks and constraints as far as possible, while preserving the best chances for pregnancy and birth. A better evaluation of embryo characteristics and of the woman's fertility can reduce the required number of transferred embryos and the multiple pregnancy rate, without reducing the chances of procreation. The main improvements that are likely to occur in the near future are the use of milder hormone treatments to stimulate ovarian function, better selection of functional gametes, and refinement of fertilization and embryo culture conditions. Furthermore, micromethods to measure the embryo's metabolic and developmental capacities should significantly improve the results of ART and infertility treatment.

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos.

BACKGROUND: In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. RESULTS: We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. CONCLUSION: Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

[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.

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm.

BACKGROUND: The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilized oocytes. RESULTS: The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilized oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilized oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilized oocytes (61.6 +/- 6.2% vs 60.7 +/- 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilized oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. CONCLUSION: The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans.