Seminal cell-free DNA and sperm characteristic's: An added biomarker for male infertility investigation.

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.

Various Genital and Reproductive Phenotypes in 46,XX/46,XY Chimeras.

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.

Serum antimüllerian hormone levels are independently related to miscarriage rates after in vitro fertilization-embryo transfer.

OBJECTIVE: To investigate whether serum antimüllerian 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.

Effect of Gonadotropin Types and Indications on Homologous Intrauterine Insemination Success: A Study from 1251 Cycles and a Review of the Literature.

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 <5 × 106 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.

High body mass index has a deleterious effect on semen parameters except morphology: results from a large cohort study.

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/m2) to extreme obesity (>40 kg/m2). 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.

How to overcome male infertility after 40: Influence of paternal age on fertility.

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 deoxyribonucleic acid damage in normozoospermic men is related to age and sperm progressive motility.

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.

Management of infertility in women over 40.

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.

Which isolated sperm abnormality is most related to sperm DNA damage in men presenting for infertility evaluation.

BACKGROUND: 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. OBJECTIVE: To evaluate sperm DNA damage in a large cohort of infertile men with isolated sperm defects. DESIGN, SETTING AND PARTICIPANTS: Retrospective study of 1084 consecutive, non-azoospermic infertile men with an isolated sperm defect: isolated oligozoospermia (iOligo), isolated asthenozoospermia (iAstheno) or isolated teratozoospermia (iTerato). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: 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. RESULTS AND LIMITATIONS: 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). CONCLUSIONS: 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.

Emerging molecular methods for male infertility investigation.

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.

Oxidative stress and fertility: incorrect assumptions and ineffective solutions?

One 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.

Carnitine content in the follicular fluid and expression of the enzymes involved in beta oxidation in oocytes and cumulus cells.

PURPOSE: The purpose of this study is to study lipid metabolism in oocytes and embryos that is a neglected parameter in human IVF. METHODS: We have tested the total carnitine content (TC) in the follicular fluid of 278 patients (217 non pregnant, 61 pregnant) undergoing IVF. RESULTS: 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. CONCLUSIONS: The addition of carnitine in oocyte maturation and embryo culture media should not be overlooked.

Sperm vacuoles are linked to capacitation and acrosomal status.

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.

Sperm transcriptome profiling in oligozoospermia.

PURPOSE: Investigate in what extent sperm transcriptome of infertile men is different from that of fertile individuals. METHODS: 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. RESULT(S): 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. CONCLUSION(S): 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.

Polymorphisms in MTHFR and MTRR genes associated with blood plasma homocysteine concentration and sperm counts.

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.

Malonaldehyde formation and DNA fragmentation: two independent sperm decays linked to reactive oxygen species.

Malondialdehyde (MDA), a product involved in membrane lipid peroxidation, was dosed in the sperm of 163 patients who had consulted the clinic regarding hypofertility. We attempted to determine if there was correlation between MDA content, sperm World Health Organization parameters and DNA fragmentation that results mainly from reactive oxygen species assaults. We found that no correlation could be established; however MDA and sperm decondensation were shown to be significantly linked. The impact of membrane polyunsaturated fatty acids and the role of phospholipid hydroperoxide glutathione peroxidase are discussed.

Paternal age and sperm DNA decay: discrepancy between chromomycin and aniline blue staining.

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.

Correlation between DNA damage and sperm parameters: a prospective study of 1,633 patients.

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.

Effect of maternal and paternal age on pregnancy and miscarriage rates after intrauterine insemination.

More than 17,000 intrauterine insemination (lUI) 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.

Comparison of sequential cytomegalovirus isolates in a patient with lymphoma and failing antiviral therapy.

BACKGROUND: Long-term anti-cytomegalovirus (CMV) treatments in immunocompromised patients are hampered by resistance to antiviral drugs. Longitudinal changes in the resistance genotype may depend on changes in selective pressure and the complexity of CMV isolates. OBJECTIVE: To evaluate longitudinal changes in the CMV resistance genotype and phenotype along with strain-specific variability in a patient with non-Hodgkin's lymphoma in whom successive anti-CMV treatments failed. STUDY DESIGN: The resistance phenotype and genotype of seven CMV isolates collected from one patient during a 2-year follow-up period were retrospectively analysed. In parallel, we used glycoprotein B (gB) genotyping, and a- and UL10-13-sequence analysis to study CMV interstrain variability. RESULTS: The patient was infected by at least three CMV strains plus variants of the parental strains. Resistance to ganciclovir, cidofovir and foscarnet was successively detected during the follow-up period. UL97 protein kinase changes responsible for resistance to ganciclovir were initially detected at residues 591 and 592, and then at position 594. Decreased sensitivity to foscarnet coincided with the appearance of amino acid substitution N495K in DNA polymerase, whereas cross-resistance to ganciclovir and cidofovir was due to the L501I substitution. CONCLUSIONS: The CMV isolates obtained from our patient were complex mixtures of strains. Changes in resistance genotypes depended on resistance selective pressure and were not linked to interstrain variation.