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Cell-free DNA (Cf-DNA) fragments may constitute an easy-to-measure molecular tool for guiding the choice of care provided to infertile couples who benefit assisted reproductive technology (ART) programmes. Data on Cf-DNA levels in the seminal plasma of men with sperm alterations are scarce. The objective of the present study was to quantify the presence of Cf-DNA in semen by using a quantitative real-time PCR. We compared men with abnormal sperm characteristics (n = 21) with normospermic controls (n = 21). The PCR assay evidenced significantly higher mean Cf-DNA levels in patients with sperm abnormalities than in controls (2.09 versus 1.18 µg/ml, respectively; p = .0003). The Cf-DNA levels were notably higher in men with azoospermia (3.65 µg/ml, versus 1.34 µg/ml in matched controls; p = .03) and men with teratozoospermia (1.80 µg/ml, versus 1.29 µg/ml in matched controls; p = .008). Our data report a significant association between elevated Cf-DNA levels and sperm abnormalities. These results may open up new diagnostic and prognostic perspectives in male infertility.

Tetragametic chimeras are due to the fusion of 2 different zygotes after fertilization. When occurring between embryos of different chromosomal sex, the phenotype ranges from fertile individuals to infertile patients and even to patients with variations in sex development. Here, we report 3 new cases of XX/XY chimeras, one in a young boy carrying an abnormal gonad which turned out to be an ovary and 2 in phenotypically normal infertile men, one of whom had been diagnosed previously as a XX-SRY negative male. These cases highlight the importance of combining several cytogenetic and molecular techniques on different tissues for a proper diagnosis and an appropriate prognosis.

Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART.

Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity.

International audience; Background: Proper folliculogenesis is fundamental to obtain a competent oocyte that, once fertilized, can support the acquisition of embryo developmental competence and pregnancy. MicroRNAs (miRNAs) are crucial regulators of folliculogenesis, which are expressed in the cumulus-oocyte complex and in granulosa cells and some can also be found in the bloodstream. These circulating miRNAs are intensively studied and used as diagnostic/prognostic markers of many diseases, including gynecological and pregnancy disorders. In addition, serum contains small amounts of cell-free DNA (cfDNA), presumably resulting from the release of genetic material from apoptotic/necrotic cells. The quantification of nucleic acids in serum samples could be used as a diagnostic tool for female infertility.Methods: An overview of the published literature on miRNAs, and particularly on the use of circulating miRNAs and cfDNA as non-invasive biomarkers of gynecological diseases, was performed (up to January 2014).Results: In the past decade, cell-free nucleic acids have been studied for potential use as biomarkers in many diseases, particularly in gynecological cancers, ovarian and endometrial disorders, as well as in pregnancy-related pathologies and fetal aneuploidy. The data strongly suggest that the concentration of cell-free nucleic acids in serum from IVF patients or in embryo culture medium could be related to the ovarian hormone status and embryo quality, respectively, and be used as a non-invasive biomarker of IVF outcome.Conclusions: The profiling of circulating nucleic acids, such as miRNAs and cfDNA, opens new perspectives for the diagnosis/prognosis of ovarian disorders and for the prediction of IVF outcomes, namely (embryo quality and pregnancy).

Objective To evaluate sperm DNA fragmentation in normozoospermic male partners of couples undergoing infertility evaluation. Design Retrospective cohort study. Setting Clinical andrology laboratory. Patient(s) A total of 1,974 consecutive normozoospermic men selected from a larger cohort of 4,345 consecutive, nonazoospermic men presenting for infertility evaluation. Intervention(s) None. Main Outcome Measure(s) Clinical parameters, conventional semen parameters, and sperm DNA fragmentation assessed by flow cytometry-based TUNEL assay and reported as percent sperm DNA fragmentation (%SDF). Result(s) The mean (±SD) %SDF and the proportion of men with high %SDF (>30%) were significantly lower in the normozoospermic compared with the entire cohort of 4,345 evaluable infertile men (17.6% ± 10.1% vs. 20.7% ± 12.4% and 11% vs. 20%, respectively). In the group of 1,974 normozoospermic men, %SDF was positively correlated with paternal age ( r = 0.17) and inversely correlated with progressive motility ( r = −0.26). In the subset of normozoospermic men with sperm parameters above the 50th percentile (≥73 × 10 6 sperm/mL, ≥55% progressive motility, and ≥14% normal forms, World Health Organization 2010 guidelines), 5% (4 of 83) had elevated %SDF (>30%). Conclusion(s) In this large cohort of normozoospermic men presenting for infertility evaluation, DNA fragmentation level is related to sperm motility and paternal age, and 11% of these men have high levels of sperm DNA fragmentation. Furthermore, the data indicate that a nonnegligible proportion (5%) of normozoospermic men with high-normal sperm parameters may also have significant sperm DNA fragmentation.

a b s t r a c t The recent trend toward delayed parenthood raises major safety concerns because of the adverse effects of aging on couple fertility. Studies have demonstrated that aging clearly affects female fertility, but can also affect male fertility. Although several theories have been proposed, the exact mechanisms responsible for the observed age-related decline in male fertility remain to be elucidated. It has been shown that advanced paternal age (PA) is associated with reduced semen volume as well as, reduced sperm count, motility and morphology. Recent studies have also reported that paternal aging is associated with a significant increase in the prevalence of both genomic and epigenomic sperm defects. In the context of natural and intrauterine insemination (IUI) conception, advanced paternal age has been associated with lower pregnancy rates and increased rates of spontaneous abortion (independent of maternal age). In IVF and oocyte donation programs, a significant decrease in late blastocyst development has been seen in those cycles using spermatozoa of men older than 55. However, no significant relationship between paternal age and IVF or ICSI pregnancy rates has been observed. Although there are no treatments that can fully restore the age-related decline in male fertility, various measures have been shown to optimize male fertility potential. Specific therapies (e.g. varicocelectomy) and lifestyle changes (e.g. dietary antioxidant supplements) may help minimize some of the age-related deleterious effects on spermatogenesis, such as, oxidative stress and endocrine abnormalities.

Sperm DNA damage is common in infertile men and is associated with poor semen parameters but the impact of an isolated sperm abnormality on sperm DNA damage has not been studied.To evaluate sperm DNA damage in a large cohort of infertile men with isolated sperm defects.Retrospective study of 1084 consecutive, non-azoospermic infertile men with an isolated sperm defect: isolated oligozoospermia (iOligo), isolated asthenozoospermia (iAstheno) or isolated teratozoospermia (iTerato).We examined and compared clinical parameters, conventional semen parameters and %sperm DNA fragmentation (%SDF, assessed by flow cytometry-based Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay) in the three groups of men.The mean (±SD) %SDF was significantly higher in the iAstheno compared to the iOligo and iTerato groups (25.0 ± 14.0 vs. 19.2 ± 11.6 and 20.7 ± 12.1 %, respectively, P 0.0001). Similarly, the proportion of men with high %SDF (30 %) was significantly higher in the iAstheno compared to the iOligo and iTerato groups (31 % vs. 18 % and 19 %, respectively, P 0.0001). In the group of 713 men with iAstheno, %SDF was positively correlated with paternal age (r = 0.20, P 0.0001) and inversely correlated with %progressive motility (r = -0.18, P 0.0001). In the subset of 218 men with iTerato, %SDF was also positively correlated with paternal age (r = 0.15, P = 0.018) and inversely correlated with %progressive motility (r = -0.26, P = 0.0001).In this large cohort of infertile men with isolated sperm abnormalities, we have found that the sperm DNA fragmentation level is highest in the men with sperm motility defects and that 31 % of these men have high levels of sperm DNA fragmentation. The data indicate that poor motility is the sperm parameter abnormality most closely related to sperm DNA damage.

Objective To investigate whether serum antimullerian hormone (AMH) levels are independently related to miscarriage rates after in vitro fertilization–embryo transfer (IVF-ET). Design Cohort study. Setting University-affiliated IVF-ET center. Patient(s) A total of 1,060 patients who attained a clinical pregnancy after IVF-ET. Interventions(s) Centralized serum AMH measurements were performed within the 12 months before IVF-ET. Binary logistic regression was used to verify whether serum AMH levels were associated with the occurrence of a miscarriage independently from confounding factors, such as age and intensity of ovarian response to controlled ovarian stimulation assessed by the number of oocytes retrieved. Main Outcome Measure(s) Miscarriage rates. Result(s) In patients displaying reduced serum AMH levels, miscarriage rates were significantly increased independently from age and the number of oocytes retrieved. Conclusion(s) The present data indicate that serum AMH levels are independently associated with the occurrence of a miscarriage after IVF-ET.

Objective. To evaluate the IUI success factors relative to controlled ovarian stimulation (COS) and infertility type, this retrospective cohort study included 1251 couples undergoing homologous IUI. Results. We achieved 13% clinical pregnancies and 11% live births. COS and infertility type do not have significant effect on IUI clinical outcomes with unstable intervention of various couples’ parameters, including the female age, the IUI attempt rank, and the sperm quality. Conclusion. Further, the COS used seemed a weak predictor for IUI success; therefore, the indications need more discussion, especially in unexplained infertility cases involving various factors. Indeed, the fourth IUI attempt, the female age over 40 years, and the total motile sperm count 6 were critical in decreasing the positive clinical outcomes of IUI. Those parameter cut-offs necessitate a larger analysis to give infertile couples more chances through IUI before carrying out other ART techniques.

Circulating or “extracellular” microRNAs (miRNAs) detected in biological fluids, could be used as potential diagnostic and prognostic biomarkers of several disease, such as cancer, gynecological and pregnancy disorders. However, their contributions in female infertility and in vitro fertilization (IVF) remain unknown. This study investigated the expression profiles of five circulating miRNAs (let-7b, miR-29a, miR-30a, miR-140 and miR-320a) in human follicular fluid from 91 women with normal ovarian reserve and 30 with polycystic ovary syndrome (PCOS) and their ability to predict IVF outcomes. The combination of FF miR-30a, miR-140 and let-7b expression levels discriminated between PCOS and normal ovarian reserve with a specificity of 83.8% and a sensitivity of 70% (area under the ROC curve, AUC = 0.83 [0.73–0.92]; p 2 (p = 0.04). Moreover, FF let-7b predicted the development of expanded blastocysts with 70% sensitivity and 64.3% specificity (AUC = 0.67 [0.54–0.79]; p = 0.02) and FF miR-29a potential to predict clinical pregnancy outcome reached 0.68 [0.55–0.79] with a sensitivity of 83.3% and a specificity of 53.5% (p = 0.01). Therefore, these miRNAs could provide new helpful biomarkers to facilitate personalized medical care during IVF.

Worldwide statistics agree that at least one out of six couples has fertility problems. If the male gamete is the origin of this problem, it is generally admitted that the oxidative stress is involved. Modern life has obviously increased fertility problems through pesticides, xenoestrogenes, endocrine disrupting chemicals involved in plastic technology such as polychlorinated bisphenyls, bisphenol A, phthalates and alkylphenols… and other cosmetic additives. An important part of these compounds increases oxidative stress, at least in part. Oxidative stress is more than probably at the origin or recurrent increasing pathologies such as endometriosis. If the oocyte is theoretically able to repair oxidative stress linked decays such as DNA fragmentation and oxidation of bases, its capacity is finite and decreasing with age. In order to decrease DNA repair charge, reducing or even avoiding the generation of DNA damages related to reactive oxygen species through consumption of antioxidants compounds is often tempting: however Reasons will be provided to break from current treatments given haphazardly in the population in the age of reproduction, as well as the potential risks of over-exposure. Furthermore recommended treatments, in relation with the new concepts in oxidative stress, will be specified.

Investigate in what extent sperm transcriptome of infertile men is different from that of fertile individuals.Semen samples were collected for determination of sperm parameters as well as for RNA isolation. Gene expression profile was investigated in spermatozoa of 8 infertile and 3 fertile men by microarray analysis using the Affymetrix Chip HG-U133 Plus 2.0.We observed up to 33-fold reduction expression of genes involved in spermatogenesis and sperm motility. Furthermore, there is an important decrease in expression of genes involved in DNA repair as well as oxidative stress regulation. In this study, we also show a striking drop in expression of histone modification genes.We found that transcription profile in germ cells of men with idiopathic infertility is different from that of fertile individuals. Interestingly, about 15% of the regulated genes (Eddy Rev Reprod 4:23-30, 1999) play a role in spermatogenesis.

Résumé Introduction L’analyse fine de la morphologie des spermatozoïdes à un grossissement de 6600 x, appelée MSOME (motile sperm organellar morphology examination) et appliquée en ICSI, a donné naissance à l’IMSI (intracytoplasmic morphologically selected sperm injection). Cette technique est proposée aux couples en échecs répétés d’implantation embryonnaire en ICSI, dans le but d’obtenir une grossesse évolutive. Matériel et méthodes L’étude concerne une cohorte observationnelle de 11535 ICSI pratiquées avec du sperme éjaculé frais, de janvier 2004 à juillet 2009. Parmi celles-ci, 2509 ont été réalisées avec IMSI. Les paramètres étudiés sont: le taux de clivage à J2 par ovocyte injecté, le taux de grossesses cliniques par ponction et le taux de fausses couches spontanées, en fonction du rang de la tentative et/ou de la qualité du sperme du bilan. Ces données ont été comparées entre l’ICSI et l’IMSI et les comparaisons ont été faites à l’aide de Chi2 et d’analyse de variance. Résultats Nous n’avons pas observé de différence significative entre l’ICSI et l’IMSI en termes de taux de clivage à J2 et taux de grossesses cliniques. Par contre, le taux de grossesses, en IMSI de rang 1, est significativement plus élevé en cas d’anomalie du sperme si on regroupe les tératozoospermies avec les oligozoospermies et les oligotératozoospermies (34,4 vs 27,1%, p = 0,02). De plus, si l’on regroupe les tératozoospermies et les oligotératozoospermies, le taux de fausses couches est plus faible en IMSI comparé à l’ICSI, de manière proche de la significativité (12,6% vs. 19,6%, p = 0,08). Conclusion En présence d’une tératozoospermie sévère, l’IMSI semble augmenter les taux de grossesses et diminuer les taux de fausses couches.

Resume Objectifs Plusieurs etudes ont indique un declin relatif dans le temps de la qualite du sperme. Dans ce contexte, 101 404 spermogrammes-spermocytogrammes, executes dans un seul laboratoire de janvier 2000 a decembre 2009, ont ete etudies retrospectivement. Les variations des parametres du sperme ont ete analysees au cours de ces dix annees. Patients et methodes Pour chaque echantillon de sperme, l’âge, la duree de l’abstinence sexuelle, le volume, pH, la concentration des spermatozoides, le pourcentage de spermatozoides mobiles, atypiques (David modifie) et l’amplitude de deplacement lateral de la tete (ALH) ont ete analyses. Pour chaque parametre, la valeur moyenne par an a ete determinee. Les tests statistiques etaient des analyses de variance et l’etablissement d’un coefficient de correlation. Resultats Les donnees ont montre une augmentation de l’âge des patients tandis que la concentration spermatique est passee de 63,5 millions/mL en 2000 a 63,6 millions/mL en 2009 avec une legere chute de 2001 a 2008. La vitalite a augmente de 67,8 % en 2000 a 73,5 % de 2009 ainsi que la mobilite globale (de 37,7 % a 39,2 %) et progressive (de 34,6 % a 36,2 %). Le pourcentage des spermatozoides atypiques est passe de 64,5 % a 84,2 % (r = 0,43, p Discussion et conclusion Contrairement a de nombreux travaux, notre etude d’une tres grande serie d’analyses de spermes n’a pas montre de reel declin des parametres du sperme selon l’OMS au cours d’une periode de dix ans, excepte pour le pourcentage de spermatozoides atypiques, lequel a considerablement augmente au cours des annees.

Resume L’analyse morphologique du spermatozoide (ou spermocytogramme) fait partie du bilan d’infertilite du couple et permet de definir la normalite d’un sperme ; cependant, la relation entre morphologie spermatique et fertilite est loin d’etre etablie. Bien que plusieurs etudes ne montrent pas de relation entre formes anormales et resultats de l’ICSI, il semble neanmoins que les taux de succes dependent de l’aspect du spermatozoide injecte. L’examen de la morphologie fine du spermatozoide (et notamment de la tete), a tres fort grossissement (de × 6600 a × 12500) (MSOME) en temps reel, permet de selectionner les plus normaux et de les injecter (IMSI). Cette selection permet d’augmenter, dans certaines situations, les taux de grossesse evolutive et d’implantation. Des criteres ultramorphologiques ont ete etablis et il semble que le plus predictif de la qualite du spermatozoide soit la presence de vacuoles dans la tete du spermatozoide. De plus, ces vacuoles semblent reliees a des alterations de l’ADN (fragmentation et/ou decondensation) ayant pour consequence des defauts du developpement embryonnaire. Pour objectiver les observations, beaucoup d’auteurs ont essaye de mettre au point des classifications et d’etablir un spermocytogramme a fort grossissement qui pourrait etre utilisable en routine et pourquoi pas dans l’avenir, remplacer le spermocytogramme.

ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.

International audience; Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART.

Objective To evaluate the influence of body mass index (BMI) on semen characteristics. Design Cohort study. Setting Single private andrology laboratory. Patient(s) All patients (n = 10,665) consulting for a semen analysis from October 9, 2010, to October 8, 2011. When analyses were repeated on the same patient, only the first was included. Intervention(s) Recording of self-reported weight and height and of semen analysis. Main Outcome Measure(s) All parameters of standard semen analysis: pH, volume, sperm concentration per mL, total sperm count per ejaculate, motility (%) within 1 hour after ejaculation (overall and progressive), viability (%), and normal sperm morphology (%). Parametric and nonparametric statistical methods were applied, and results are given either with mean ± SD, or 10th, 50th, and 90th percentiles. Result(s) Semen volume decreased from 3.3 ± 1.6 to 2.7 ± 1.6 mL when BMI increased from normal (20–25 kg/m 2 ) to extreme obesity (>40 kg/m 2 ). The same was true for semen concentration (56.4 ± 54.9 to 39.4 ± 51.0 million/mL), total sperm count (171 ± 170 to 92 ± 95 million), and progressive motility (36.9 ± 16.8% to 34.7 ± 17.1%). The percentage of cases with azoospermia and cryptozoospermia increased from 1.9% to 9.1% and from 4.7% to 15.2%, respectively. The other semen characteristics were not affected. Multivariate models including age and abstinence duration confirmed these results. Conclusion(s) In this study, on a large patient sample size, increased BMI was associated with decreased semen quality, affecting volume, concentration, and motility. The percentage of normal forms was not decreased.

Male factors account for approximately 50% of reproductive pathology. Different disorders, including urogenital and endocrine system development abnormalities, lead to testicular and gametogenesis defects. Parallely, studies have reported that somatic and germ cell genome decay are a major cause of male infertility. It has been shown that in somatic karyotype, there is a higher incidence of chromosomal aberrations in infertile men than neonatal population and significant chromosome Y microdeletion or specific gene alterations in affected spermatogenesis. Karyotyping and FISH application at somatic and germ cell levels are no longer sufficient to investigate the potential contribution of genome disorders on male infertility. A wide range of molecular methods are required for better understanding of male infertility causes. Molecular omes and omics techniques have become a great tool to investigate male infertility from chromosome to protein. This review reports different molecular tests and methods that can be offered for male infertility investigation.

Women's fertility potential is declining with age because of multiples intrinsic and extrinsic factors such as life style, oxidative stress and/or endocrine disruptors and is affecting the ability of these women to conceive naturally. This declining fertility potential and the late age of motherhood is increasing significantly the number of patients consulting infertility specialists. Different strategies of investigation and management are proposed to patients over 40 in order to overcome their infertility and improve the live birth rate in these patients. Intra Uterine Insemination (IUI) in women over 40 is associated with a low rate of ongoing pregnancy and IUI should not therefore be offered always as the first line of treatment. When the predictive factors are positive IVF/ICSI seem to be good alternatives until 43 years of age. Customized ovarian stimulation and flexible laboratory methods such as in vitro maturation (IVM), preimplantation genetic diagnosis (PGD), embryo vitrification and transfer after thawing in subsequent natural or artificial cycles can improve the success rate of ART in patients over 40. Meanwhile, oocyte and embryos donation remain good options for patient over 40 with a bad prognosis and can lead to successful ongoing pregnancies until 45 years of age. Ovarian tissue cryopreservation, oocyte vitrification at the germinal vesicle (GV) stage or metaphase II stage present a breakthrough for fertility preservation but the ideal age for starting fertility preservation is still debated as well as the minimum number of oocytes to be vitrified in order to optimize the chances of pregnancy when needed at an older age. This manuscript reports the results of our own experience from patients older than 40 in the light of the published data and discusses the different therapeutic alternatives which can be proposed to patients over 40 consulting ART centres.

STUDY QUESTION Is the presence of nuclear vacuoles really a negative parameter? SUMMARY ANSWER As sperm vacuoles are associated with acrosomal and capacitation status, they appear to be a reflection of normal sperm physiology. WHAT IS KNOWN ALREADY The selection of sperm under a high magnification has been proposed as a strategy to increase the success rates of ICSI, through a better selection of sperm for injection. The presence of vacuoles on the sperm head is said to be a negative parameter. STUDY DESIGN, SIZE, DURATION We incubated processed sperm for 90 min with two strong inducers of acrosome reaction (AR), i.e. hyaluronic acid (HA) and follicular fluid (FF) and studied the evolution of nuclear vacuoles, sperm morphology and chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHODS We tested the effect of incubating sperm samples with HA and FF for 90 min at 37°C on nuclear vacuoles. MAIN RESULTS AND THE ROLE OF CHANCE Both HA and FF strongly induce AR after 90 min, without significantly modifying sperm nuclear condensation and morphology (Bartoov's criteria). We simultaneously observed a highly significant decrease in the presence of vacuoles. LIMITATIONS, REASONS FOR CAUTION This is a descriptive study based on in vitro manipulations. WIDER IMPLICATIONS OF THE FINDINGS Although intracytoplasmic morphologically selected sperm injection may be of benefit for couples with specific treatment indications, the results of this study make it difficult to justify its large-scale application. STUDY FUNDING/COMPETING INTEREST(S) Funding was granted by Laboratoire d'Eylau, Unilabs.

SummaryOne of the most important concerns in assisted reproduction (ART), and in particular ICSI, is the quality of sperm DNA. Oxidative stress is one of the major causes of damage to DNA and attempting to reduce generation of DNA damage related to reactive oxygen species (ROS) through consumption of antioxidants is often tempting. However, current antioxidant treatments, given irrespectively of clinically quantified deficiencies, are poorly efficient, potentially detrimental and over-exposure is risky. Here we discuss new treatments in relation to present day concepts on oxidative stress. This discussion includes stimulation of endogenous anti-ROS defense i.e. glutathione synthesis and recycling of homocysteine, the epicentre of multiple ROS-linked pathologies.

The purpose of this study is to study lipid metabolism in oocytes and embryos that is a neglected parameter in human IVF.We have tested the total carnitine content (TC) in the follicular fluid of 278 patients (217 non pregnant, 61 pregnant) undergoing IVF.The follicular fluid TC is neither correlated with the circulating estradiol content in serum nor with the outcome the IVF attempt. Carnitine, through the carnitine shuttle, is a major partner in lipid beta oxidation, metabolic pathway involved in the acquisition of oocyte competence. The expression of carnitine synthesis enzymes and lipid beta oxidation was studied in cumulus cells collected at the time of ovum retrieval and in oocyte. Surprisingly the expression for carnitine synthesis is not detectable in oocytes whereas the enzymes involved in lipid beta oxidation are rather strongly expressed.The addition of carnitine in oocyte maturation and embryo culture media should not be overlooked.

Worldwide statistics agree that at least one out of six couples has fertility problems. If the male gamete is the origin of this problem, it is generally admitted that the oxidative stress is involved. Modern life has obviously increased fertility problems through pesticides, xenoestrogenes, endocrine disrupting chemicals involved in plastic technology such as polychlorinated bisphenyls, bisphenol A, phthalates and alkylphenols… and other cosmetic additives. An important part of these compounds increases oxidative stress, at least in part. Oxidative stress is more than probably at the origin or recurrent increasing pathologies such as endometriosis. If the oocyte is theoretically able to repair oxidative stress linked decays such as DNA fragmentation and oxidation of bases, its capacity is finite and decreasing with age. In order to decrease DNA repair charge, reducing or even avoiding the generation of DNA damages related to reactive oxygen species through consumption of antioxidants compounds is often tempting: however Reasons will be provided to break from current treatments given haphazardly in the population in the age of reproduction, as well as the potential risks of over-exposure. Furthermore recommended treatments, in relation with the new concepts in oxidative stress, will be specified.

Depuis quelques annees, plusieurs publications ont mis en exergue l’avantage de l’examen du sperme au fort grossissement (× 6 600), permettant l’evaluation de la morphologie du noyau spermatique et la densite de sa chromatine en temps reel avant micro-injection ovocytaire. Ce procede est communement appele motile sperm organelle morphology examination (MSOME). La technique d’ICSI est appelee intracytoplasmic morphologically selected sperm injection (IMSI). En reproduction, nous deplorons helas plus d’echecs que de succes. Les causes des echecs sont multiples, d’origine maternelle mais aussi paternelle. En effet, un sperme inadequat peut conduire au pire a un defaut de fecondation ou de developpement embryonnaire precoce et au mieux a une implantation embryonnaire suivie d’une fausse couche.

Several studies have reported a time-related decline in semen quality. In this context, 101,404 spermograms-spermocytograms performed in a single andrology laboratory from January 2000 to December 2009 were investigated retrospectively. Variations of sperm parameters were analyzed over the years.For each semen sample, age at semen collection, duration of sexual abstinence, volume of seminal fluid, pH, concentration of spermatozoa, percentages of overall motile and progressive motile sperm, percentage of morphologically abnormal spermatozoa (according to David's classification) and amplitude of lateral head displacement (ALH) were analyzed. For each parameter, the mean value per year was determined. To examine trends over time the statistical tests used were analyses of variance and correlation studies.Data showed an increasing age of patients from 36.5 years in 2000 to 37.2 years in 2009. The semen concentration was 63.5 millions/mL in 2000 and 63.6 millions/mL in 2009 with a slight fall from 2001 to 2008. The vitality continuously increased from 67.8% in 2000 to 73.5% in 2009 as well as the overall motility (from 37.7% to 39.2%) and the progressive motility (from 34.6% to 36.2%). The percentage of morphologically abnormal spermatozoa continuously increased from 64.5% to 84.2% (r=0.43, P0.001).Contrary to a variety of works, our very large study of semen analysis did not show a real decline in semen WHO parameters during a 10-year period, except for the percentage of morphologically abnormal spermatozoa, which dramatically increased over the years.

Objective To investigate the relationship between MTHFR and MTRR genetic variants with respect to both blood plasma homocysteine concentration and sperm counts. Design Polymerase chain reaction followed by specific enzymatic digestion to determine the genotype of the individuals and blood plasma homocysteine quantification by high-performance liquid chromatography. Setting Research laboratory. Patient(s) Two hundred sixty-eight men seeking infertility counseling and 254 partners of infertile women. Intervention(s) We studied three MTHFR (c.1286A→C, c.665C→T and c.203G→A) and two MTRR (c.66A→G and c.524C→T) single-nucleotide polymorphisms and characterized sperm parameters in both oligozoospermic and normospermic men. A cohort of 522 men was examined for this study. A subgroup of 103 men was constituted for quantification of Hcy levels. Main Outcome Measure(s) Semen samples were collected for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines as well as for DNA isolation. Blood samples of the corresponding individuals were obtained to quantify plasma homocysteine levels. Result(s) We did not observe a relationship between homocysteinemia and sperm counts. The MTHFR c.665C→T variant is associated with mild hyperhomocysteinemia in blood plasma in the TT homozygous state. Conclusion(s) No association was found between MTHFR / MTRR genetic variants and sperm counts. Although no association was observed with reduced sperm counts, the MTHFR 665TT genotype is associated with a significant increase in blood plasma homocysteine levels.

The effect of paternal age on sperm DNA fragmentation and decondensation was determined in a retrospective study involving 1769 patients. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay was used to assess fragmentation, and DNA decondensation was measured with either chromomycin or aniline blue staining. The impact of atypical forms was also analysed. DNA fragmentation increases with age, but is independent of the percentage of atypical forms. Both staining techniques revealed a negative correlation between the quality of sperm packaging and the percentage of atypical forms. Decondensation increases with increasing age and fragmentation when measured with chromomycin; however, an inverse relationship is observed when testing is performed using aniline blue. These observations are discussed in relation to the specificity of the dyes, the deposition of protamines and the impact of age and reactive oxygen species on protamine cross-linking.

Until now, the morphological sperm analysis (spermocytogram) allows to define sperm normality, but the relationship between sperm morphology and fertility is not yet assessed. Although several studies do not report any relationship between abnormal sperm morphology and ICSI results, nevertheless, the success rate of ICSI sems to be dependent on injected sperm morphological aspect. Detailed morphological sperm examination (especially sperm head) at high magnification (from x 6600 to x 12500) (MSOME) in real time allows to select the best spermatozoa before oocyte injection (IMSI). In some cases, implantation and ongoing pregnancy rates were improved with this sperm selection method. Ultramorphologic criteria were established and the most predictive factor of sperm quality is the presence of vacuoles in the sperm head. Those vacuoles appear to be related to DNA damage (fragmentation and/or denaturation) and affect embryo development. To standardize those observations, several authors tried to establish sperm MSOME classifications in order to be used in routine and to replace the conventional spermocytogram in the next future.

More than 17,000 intrauterine insemination (IUI) cycles were analysed retrospectively with respect to outcome according to differing aetiologies of infertility. The quantity and motility of spermatozoa in the final preparation used for insemination had a positive effect on the outcome, as classically observed in the past. It was found that advanced maternal age had a negative effect on the pregnancy rate and was associated with increased miscarriage rate. More interestingly, an exactly parallel effect was found for paternal age. The impact of increased age on necrospermia and sperm DNA structure is discussed as a probable direct cause of this paternal effect.

ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.

Objective To investigate DNA fragmentation by using terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling in relation to World Health Organization parameters and computer-aided sperm analysis (CASA) in sperm to determine the possibility of obtaining a correlation among CASA parameters, sperm morphology, and DNA fragmentation. Design Sperm analysis according to World Health Organization parameters, terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) for sperm DNA fragmentation, and CASA for sperm movement. Prospective study. Setting All the patients were under clinical management, consulting for hypofertility at a fertility center in France. Patient(s) One thousand six hundred thirty-three men who were referred for infertility investigation, including a complete sperm analysis. Intervention(s) Sperm analysis and DNA damage testing. Main Outcome Measure(s) Sperm morphology, DNA fragmentation, and movement characteristics. Result(s) One third of the patients had a TUNEL rate of >30%. Analysis of the 21 semen parameters tested revealed that 7 of them were significantly correlated with the TUNEL results. Conclusion(s) World Health Organization sperm parameters and DNA damage are complementary, rather than strongly linked. This should be considered to more fully understand the paternal contribution in assisted reproductive technologies failures.