P. Jouannet

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

BackgroundIn 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.ResultsWe 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.ConclusionCompared 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.

In vitro fertilization and embryo culture strongly impact the placental transcriptome in the mouse model.

Background Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. Methodology/Principal Findings Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. Conclusions For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.

Detection of HIV-1 in seminal plasma and seminal cells of HIV-1 seropositive men

It is of critical importance to precisely understand the modalities of HIV presence in semen, especially with regard to procreation. In this study, paired blood and semen samples from 31 human immunodeficiency virus type 1 (HIV-1)-positive men were assessed for cell-free HIV-RNA load in blood plasma (BP) and seminal plasma (SP), and for detection of HIV by culture, PCR and RT-PCR in semen cellular fractions separated by centrifugation on Percoll gradient. HIV-RNA was detected in 94% of BP and 84% of SP samples. For 11 men (35%), HIV-RNA load in SP was equal or superior to that observed in blood. HIV-DNA presence was demonstrated (either by PCR or culture positivity) in 39% of the non-spermatozoal cells (NSC)-enriched fractions, and in one Percoll-selected sperm pellet. HIV-RNA was detected in 17% (4/23) of the sperm pellets. This positivity was associated with an HIV-RNA load in SP equal or superior to the HIV-RNA load in blood, a high rate of HIV-DNA detection in the NSC fraction, and a low CD4+ cell count. In such conditions, a significant viral production inside the genital tract is more likely to be present, and a close association between HIV and gametes might occur. Assisted procreation with selected spermatozoa should be preceded by accurate assessments of viral presence in blood, SP and semen cellular fractions.

Epigenetic disorders and male subfertility

OBJECTIVEnTo provide a link between epigenetics and male subfertility at the DNA, histone-protamine, and RNA levels and its consequences on fertilization and embryo development.nnnDESIGNnReview of the relevant literature.nnnSETTINGnUniversity-based clinical and research laboratories.nnnPATIENT(S)nFertile and infertile men.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nCritical review of the literature.nnnRESULT(S)nEpigenetic 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.nnnCONCLUSION(S)nThe 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.

Clinical data and parenthood of 63 infertile and Y-microdeleted men

OBJECTIVEnTo collect follow-up data for infertile men with Y microdeletion.nnnDESIGNnRetrospective, observational survey.nnnSETTINGnMulticenter IVF units associated with genetics laboratories.nnnPATIENT(S)nSixty-three patients with Y microdeletion.nnnINTERVENTION(S)nKaryotype analysis, Y microdeletion screening, and assisted reproductive technology.nnnMAIN OUTCOME MEASURESnMedical history, karyotype, nature of the AZF deletion, semen parameters, testis biopsy results, choice of assisted reproductive technology, and results of intracytoplasmic sperm injection (ICSI).nnnRESULTSnAbnormal karyotypes were found in 8 men (12.7%), who were azoospermic except 1. Of these 8 men, 5 presented a combined AZFb+c deletion, and 3 had a deletion in AZFc only. Most men (39 of 63) were azoospermic, 3 were cryptoazoospermic, and 19 had extreme oligozoospermia (sperm concentration </=1.10(6)/mL). Sperm concentration above 1.10(6)/mL was found for 2 men (3.1%). A testis biopsy was performed in 27 azoospermic men, resulting in positive sperm extraction in 6 cases. To date, 42 ICSI cycles with either testicular (n = 5) or ejaculated spermatozoa (n = 37) have been carried out in 23 couples with male partners with AZFc deletion. Eighteen clinical pregnancies were obtained, leading to the birth of 14 babies. Donor insemination had been chosen by 28 couples, leading to the birth of 9 children.nnnCONCLUSIONnKaryotype analysis should be systematically performed in Y microdeleted men. Intracytoplasmic sperm injection can be offered to half of AZFc-deleted patients, providing real opportunities to have a child.

Cumulative results including obstetrical and neonatal outcome of fresh and frozen-thawed cycles in elective single versus double fresh embryo transfers

OBJECTIVEnTo assess the efficacy of elective single embryo transfer (e-SET) compared to a double embryo transfer (DET) in a selected population including obstetrical and neonatal outcome of fresh and frozen-thawed cycles.nnnDESIGNnProspective nonrandomized study.nnnSETTINGnDepartment of reproductive medicine.nnnPATIENT(S)nElective single embryo transfer was proposed to women < 36 years with adequate ovarian function, in their first or second IVF or intracytoplasmic sperm injection (ICSI) attempt with ejaculated sperm, with at least 4 mature oocytes and 2 fertilized top quality embryos. Patients who refused e-SET had two top embryos transferred (DET group).nnnINTERVENTION(S)nMedical management and IVF-ICSI.nnnMAIN OUTCOME MEASURE(S)nCumulative delivery rate, twin delivery rate, obstetrical and neonatal outcome.nnnRESULT(S)nAccording to patients decision, 53 women had an e-SET and 98 a DET. The cumulative delivery rate per patient was 54.7% in the e-SET group and 49.0% in the DET group (P>0.05). Twin delivery rate was significantly different between the two groups (3.5% versus 37.5% respectively, P<0.05). Neonatal outcome in twins resulting from IVF-ICSI was found to be poorer than in singletons, considering the mean gestational age, mode of delivery, birthweight, and risk of neonatal intensive care unit admission for the infants.nnnCONCLUSION(S)nIn a selected population, the elective transfer of one embryo with high implantation potential helped to avoid twin pregnancies without decreasing delivery rate.

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

STUDY QUESTIONnCan 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?nnnSUMMARY ANSWERnA basic checklist for authors, reviewers and editors has been developed and is presented.nnnWHAT IS KNOWN ALREADYnLaboratory 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.nnnSTUDY DESIGN, SIZE, DURATIONnAfter 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.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnN/A.nnnMAIN RESULTS AND THE ROLE OF CHANCEnA 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.nnnLIMITATIONS, REASONS FOR CAUTIONnGuidelines 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.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe 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.nnnSTUDY FUNDING/COMPETING INTERESTSnNo funding was obtained for this work. The authors have no competing interests in relation to the present publication and checklist.nnnTRIAL REGISTRATION NUMBERnN/A.

Another look at human sperm morphology

STUDY QUESTIONnCan a standardized assessment of abnormal human sperm morphology provide additional useful information by identifying men with more severe disturbances in different types of abnormalities?nnnSUMMARY ANSWERnDefinition-based categorization of sperm head, midpiece and tail defects has shown how differently these abnormalities are distributed in fertile men and other groups of men, thus providing high and low thresholds, a starting point for diagnosis or research purposes.nnnWHAT IS KNOWN ALREADYnSeveral recent studies have reported indisputable genetic origins for various sperm defects. A few studies have also identified associations between environmental factors and low percentages of morphologically normal spermatozoa. Nevertheless, with the exception of rare situations in which the vast majority of spermatozoa have specific, easily characterized defects, such as globozoospermia, little attention has been paid to the description and precise quantification of human sperm abnormalities. The lack of standardization in the phenotyping of sperm morphological defects by conventional microscopy is a limiting factor for diagnosis and for intra- or inter-observer or centre consistency in studies investigating the causal factors and possible functional consequences of the abnormalities detected. There are currently no baseline data for abnormalities of sperm morphology based on a standardized classification, in the general population, among fertile or other groups of men.nnnSTUDY DESIGN, SIZE, DURATIONnThis study is based on detailed sperm abnormality datasets acquired by a standardized classification method, from several groups of men, over the same 5-year period.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWe studied cross-sectional data from fertile men (n = 926), male partners from infertile couples (n = 1747) and testicular cancer patients (n = 239). We used a standardized classification to analyse Shorr-stained slides, taking into account all the abnormalities encountered.nnnMAIN RESULTS AND THE ROLE OF CHANCEnMost sperm defects were significantly more frequent in infertile than in fertile men, with 20-30% of infertile men having frequencies of abnormalities above the 95th percentile in fertile men for 9 out of the 15 categories of abnormalities. Interestingly, several head abnormalities were significantly more frequent in patients with testicular cancer than in infertile men, highlighting the particular impact of this condition on sperm morphogenesis. We used the 95th percentile in fertile men as the lower threshold and the 99th percentile in infertile men as an extreme upper threshold, for the classification of morphological abnormality frequencies into three levels: low, intermediate and high. The assessment of several semen samples, with or without a genetic background, for abnormal sperm morphology, based on the percentage of normal spermatozoa, a teratozoospermia index, and the detailed profile of abnormalities categorized according to the three levels proposed, has highlighted the value of detailed phenotyping for diagnosis and research purposes.nnnLIMITATIONS, REASONS FOR CAUTIONnThe thresholds proposed for the various categories of sperm abnormality should be considered relative rather than absolute, owing to the known sampling error related to the limited number of spermatozoa assessed per sample, or when studying the general population or populations from regions other than Western Europe. The standardized assessment of abnormal sperm morphology requires time and experience. We therefore suggest that this assessment is carried out during a first andrological check-up or for epidemiological or research studies, rather than in the routine management of infertile couples for assisted reproductive technologies.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe study design used for the fertile group of men was similar to that previously used for the WHO reference values, providing a rationale for considering the 95th percentile in fertile men as the level below which abnormalities may be considered to occur at a frequency representing random background variations of a normal spermiogenesis process. The crude frequencies obtained, and the three levels of abnormality frequency proposed for each standardized category of sperm defect, provide baseline data useful for diagnosis and a starting point for future studies aiming to identify associations with genetic or environmental factors.nnnSTUDY FUNDING/COMPETING INTERESTSnPart of this study was supported by contract BMH4-CT96-0314 from the European Union. The authors have no competing interests to declare.

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

BackgroundThe 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 unfertilised oocytes.ResultsThe hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised 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.ConclusionThe 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.

Cardiovascular findings in women suffering from Turner syndrome requesting oocyte donation

BACKGROUNDnThrough oocyte donation (OD), women with Turner syndrome (TS) may achieve motherhood. However, this population has a high prevalence of cardiac malformations and carry a risk for aortic dissection that is increased by pregnancy. Until recently, the necessity for a specialized cardiac evaluation before pregnancy was underestimated as was the need for follow-up through adulthood. The aim of this study was to evaluate the follow-up (mainly cardiovascular) of women with TS requesting OD.nnnMETHODSnDisease monitoring since diagnosis and prior cardiac evaluations conducted out of our centre were assessed in 25 women with TS who requested OD. New cardiac evaluations using echocardiography and magnetic resonance imaging were performed by our specialized cardiologist in 18 of these patients.nnnRESULTSnWe observed that the medical follow-up of women with TS was often deficient throughout adulthood. Most of the prior cardiac evaluations performed by cardiologists not accustomed to women with TS, either before (n = 8) or when starting OD (n = 12), were considered normal. However, when revaluated by a cardiologist who is familiar with TS, seven women were diagnosed with a bicuspid aortic valve and thus excluded from OD. In addition, when appropriate screening was conducted by our referent cardiologist before OD no cardiac complication was observed during pregnancy or delivery.nnnCONCLUSIONSnCareful follow-up, including cardiac evaluation, should be recommended for women diagnosed with TS, before and after puberty. Moreover, assessment of cardiovascular parameters by a cardiologist familiar with TS should be routinely repeated before undertaking OD.