The impact of ejaculatory abstinence on semen analysis parameters: a systematic review.

OBJECTIVE: The aim of this study was to evaluate recent publications and determine the impact of ejaculatory abstinence on semen analysis parameters as well as fertility outcomes. METHODS: This was a systematic review of 28 recent publications. The focus of this study was the impact of abstinence on semen parameters and fertility outcomes in papers published since the year 2000. The specific parameters evaluated were volume, sperm count, motility, morphology, pH, DNA fragmentation rate, viability, and pregnancy or fertilization rates following assisted reproduction. RESULTS: Twenty-eight recent publications met inclusion criteria. Analysis of publications showed that longer abstinence is associated with increases in semen volume and sperm count. Studies evaluating the effect of abstinence on motility, morphology, and DNA fragmentation rates are contradictory and inconclusive, although a trend appears to exist toward improvements in semen parameters with shorter abstinence. Semen pH was unaffected by abstinence. The majority of publications found no difference in rates of viability with varying abstinence times, although total number of viable sperm increases with increasing abstinence. Some studies evaluating the impact of ejaculatory abstinence on intrauterine insemination (IUI), intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF) demonstrated an association between short abstinence and improved outcomes. CONCLUSIONS: The impact of abstinence on sperm quality is complex. While certain semen parameters improve with longer abstinence, others appear to improve with shorter abstinence. No clear recommendations can be made regarding ideal abstinence due to the conflicting nature of current evidence. Going forward, more research is needed to evaluate the impact of abstinence on pregnancy and fertilization rates.

Phase II study of avatrombopag in thrombocytopenic patients with cirrhosis undergoing an elective procedure.

BACKGROUND & AIMS: This is a phase II multicentre study to investigate the efficacy and safety of avatrombopag (E5501), an investigational second-generation thrombopoietin receptor agonist, administered one week prior to elective procedures in patients with thrombocytopenia secondary to cirrhosis. METHODS: Adults with cirrhosis and platelet counts ⩾10 to ⩽58×10(9)/L were randomized to placebo or avatrombopag in two sequential cohorts. Cohort A: placebo vs. one of 3 different doses (100mg loading dose followed by 20, 40, or 80 mg/day on days 2-7) of a first-generation avatrombopag formulation. Cohort B: placebo vs. one of 2 different doses (80 mg loading dose followed by 10 mg/day for days 2-7, or 20mg/day for days 2-4) of a second-generation avatrombopag formulation. Primary end point was achievement of a platelet increase of ⩾20×10(9)/L from baseline and >50×10(9)/L at least once during days 4-8. RESULTS: A total of 130 patients were randomized: 93 patients (51, cohort A; 42, cohort B) to avatrombopag and 37 (16, cohort A; 21 cohort B) to placebo. The primary end point was achieved by 49.0% of treated patients in cohort A and 47.6% in cohort B compared to 6.3% and 9.5% of controls; a dose response was seen. Each avatrombopag regimen had a higher proportion of responders compared with their respective cohort placebo arms (p<0.01), except for the 100/40 mg group in cohort A (p=0.17). The most common adverse events were nausea, fatigue, and headache. One patient in the (100/80) avatrombopag group, without a Doppler assessment at screening was diagnosed with portal vein thrombosis during post-treatment follow-up. CONCLUSIONS: In this study avatrombopag was generally well-tolerated and increased platelet counts in patients with cirrhosis undergoing elective invasive procedures.

Altered Epigenetic Profiles in the Placenta of Preeclamptic and Intrauterine Growth Restriction Patients.

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are placental pathologies known to complicate pregnancy and cause neonatal disorders. To date, there is a limited number of studies on the genetic similarity of these conditions. DNA methylation is a heritable epigenetic process that can regulate placental development. Our objective was to identify methylation patterns in placental DNA from normal, PE and IUGR-affected pregnancies. DNA was extracted, and bisulfite was converted, prior to being hybridized for the methylation array. Methylation data were SWAN normalized and differently methylated regions were identified using applications within the USEQ program. UCSC's Genome browser and Stanford's GREAT analysis were used to identify gene promoters. The commonality among affected genes was confirmed by Western blot. We observed nine significantly hypomethylated regions, two being significantly hypomethylated for both PE and IGUR. Western blot confirmed differential protein expression of commonly regulated genes. We conclude that despite the uniqueness of methylation profiles for PE and IUGR, the similarity of some methylation alterations in pathologies could explain the clinical similarities observed with these obstetric complications. These results also provide insight into the genetic similarity between PE and IUGR and suggest possible gene candidates plausibly involved in the onset of both conditions.

Epigenetic determinants of reproductive potential augment the predictive ability of the semen analysis.

OBJECTIVE: To investigate the power of DNA methylation variability in sperm cells in assessing male fertility potential. DESIGN: Retrospective cohort. SETTING: Fertility care centers. PATIENTS: Male patients seeking infertility treatment and fertile male sperm donors. INTERVENTION: None. MAIN OUTCOME MEASURES: Sperm DNA methylation data from 43 fertile sperm donors were analyzed and compared with the data from 1344 men seeking fertility assessment or treatment. Methylation at gene promoters with the least variable methylation in fertile patients was used to create 3 categories of promoter dysregulation in the infertility treatment cohort: poor, average, and excellent sperm quality. RESULTS: After controlling for female factors, there were significant differences in intrauterine insemination pregnancy and live birth outcomes between the poor and excellent groups across a cumulative average of 2-3 cycles: 19.4% vs. 51.7% (P=.008) and 19.4% vs. 44.8% (P=.03), respectively. Live birth outcomes from in vitro fertilization, primarily with intracytoplasmic sperm injection, were not found to be significantly different among any of the 3 groups. CONCLUSION: Methylation variability in a panel of 1233 gene promoters could augment the predictive ability of semen analysis and be a reliable biomarker for assessing intrauterine insemination outcomes. In vitro fertilization with intracytoplasmic sperm injection appears to overcome high levels of epigenetic instability in sperm.

Tissue-specific DNA methylation variability and its potential clinical value.

Complex diseases have multifactorial etiologies making actionable diagnostic biomarkers difficult to identify. Diagnostic research must expand beyond single or a handful of genetic or epigenetic targets for complex disease and explore a broader system of biological pathways. With the objective to develop a diagnostic tool designed to analyze a comprehensive network of epigenetic profiles in complex diseases, we used publicly available DNA methylation data from over 2,400 samples representing 20 cell types and various diseases. This tool, rather than detecting differentially methylated regions at specific genes, measures the intra-individual methylation variability within gene promoters to identify global shifts away from healthy regulatory states. To assess this new approach, we explored three distinct questions: 1) Are profiles of epigenetic variability tissue-specific? 2) Do diseased tissues exhibit altered epigenetic variability compared to normal tissue? 3) Can epigenetic variability be detected in complex disease? Unsupervised clustering established that global epigenetic variability in promoter regions is tissue-specific and promoter regions that are the most epigenetically stable in a specific tissue are associated with genes known to be essential for its function. Furthermore, analysis of epigenetic variability in these most stable regions distinguishes between diseased and normal tissue in multiple complex diseases. Finally, we demonstrate the clinical utility of this new tool in the assessment of a multifactorial condition, male infertility. We show that epigenetic variability in purified sperm is correlated with live birth outcomes in couples undergoing intrauterine insemination (IUI), a common fertility procedure. Men with the least epigenetically variable promoters were almost twice as likely to father a child than men with the greatest number of epigenetically variable promoters. Interestingly, no such difference was identified in men undergoing in vitro fertilization (IVF), another common fertility procedure, suggesting this as a treatment to overcome higher levels of epigenetic variability when trying to conceive.

Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1.

We assessed the efficacy and safety of 10-d monotherapy with the orally administered CCR5 antagonist maraviroc in 63 HIV-1-positive individuals prescreened for the absence of CXCR4-using virus. Maximum reduction in viral load occurred at a median of 10-15 d, with a mean reduction of >or=1.6 log(10) copies/ml at all twice daily doses >or=100 mg. These results provide proof of concept that CCR5 antagonism is a viable antiretroviral therapeutic approach.

Tissue specific age acceleration patterns in the sperm of oligozoospermic men.

To determine if disease can modify aging patterns in an affected tissue without altering the aging patterns of other tissues, blood and semen of individuals with oligozoospermia (n = 10) were compared to the blood and semen of individuals with normozoospermia (n = 24). DNA methylation data was obtained via Illumina's 850 K array. The Horvath and Jenkins age calculators were then utilized to predict the epigenetic age of blood and sperm. Epigenetic age of sperm was approximated using germ-line age differential (GLAD) values. Using nonpaired t-tests, it was found that sperm of oligozoospermic men (mean GLAD score of 0.078) were predicted to be significantly older than the sperm of normozoospermic men (mean GLAD score of -0.017), returning a p-value of 0.03. However, there was not a significant epigenetic age difference between the blood of those with oligozoospermia (mean GLAD equivalent score of -0.027) and normozoospermia (mean GLAD equivalent score of 0.048), producing a p-value of 0.20. These results lead to the conclusion that tissue specific aging is occurring in sperm of oligozoospermic individuals but not in unaffected somatic tissues (in this case, blood).

Assessment of seminal cell-free DNA as a potential contaminate in studies of human sperm DNA methylation.

BACKGROUND: Human seminal cell-free deoxyribonucleic acid (cfDNA) methylation patterns have not yet been thoroughly explored; however, recent work in mouse has suggested that some cfDNA encountered in the epididymis may contaminate DNA methylation studies assessing the mature spermatozoa. Such contamination could clearly prove to be a significant confounder, for many reasons, in epigenetic studies of male factor infertility. OBJECTIVES: To explore the nature of seminal cfDNA methylation and the likelihood that it would be retained following standard semen sample processing for epigenetic analysis. MATERIALS AND METHODS: We assessed 12 semen samples collected at Utah Fertility Center. For each sample, seminal cfDNA was isolated from the sperm pellet. The spermatozoa was split into three aliquots including one exposed to DNase to remove any additional cfDNA (termed "pure spermatozoa"), one not exposed to DNase, and one exposed to DNase but reintroduced to seminal cfDNA. We additionally assessed blood DNA as our benchmark for somatic cell DNA methylation patterns. DNA methylation was measured via Illumina's 850k array and assessed for differential regional methylation. RESULTS: Forty-six thousand three hundred fifty-two differentially methylated regions (FDR > 40) were identified between pure spermatozoa and seminal cfDNA. We found at these sites that the average DNA methylation in cfDNA always fell somewhere between the average methylation in spermatozoa and blood. We also assessed each sperm treatment groups at all 46,352 regions of interest and found no significant differences at any of these sites. DISCUSSION AND CONCLUSION: Our data suggest that seminal cfDNA is a clear mixture of both somatic and germline DNA and that cfDNA is not a contaminating feature in sperm DNA methylation studies following standard protocols in human sperm DNA extraction.

Effect of chemotherapy on the uterus of young adult cancer survivors.

Objective: To investigate the impact of chemotherapy on the uterus. Design: Cross-sectional pilot study. Setting: Single university fertility clinic. Patients: Twelve patients with a history of alkylating agent chemotherapy exposure after Hodgkin lymphoma (cancer) vs. 12 normally menstruating women (controls). Interventions: The inclusion criteria were age of 18-45 years and consent for endometrial biopsy. The exclusion criteria were the absence of the uterus, completed pelvic radiation, uterine or cervical cancer, and metastatic cancer. Each participant underwent endometrial biopsy and pelvic ultrasound. All study visits were conducted in the late proliferative phase of the menstrual cycle. Main Outcome Measures: Uterine volume, blood flow, endometrial thickness, histology, deoxyribonucleic acid methylation pattern, and relative ribonucleic acid (RNA) expression level during the same phase of the menstrual cycle. Results: In the study group, visits were conducted at a median of 31.5 (13.5-42.5) months after chemotherapy. The median uterine volume among cancer survivors was 36 (11.3-67) cm3, and that of the general population controls was 39 (13-54) cm3. On histologic examination, there were no cytologic or architectural atypia. The RNA-sequencing analysis revealed poor clustering of both control and treatment samples. However, we identified 3 differentially expressed genes on RNA-sequencing, but there was no concordance found among the differentially expressed genes and deoxyribonucleic acid methylation changes suggesting most likely false-positive results. Conclusions: Approximately 2.5 years after chemotherapy, a time at which several survivors of Hodgkin lymphoma may resume family-building, endometrial thickness and endometrial histology were not significantly affected by a history of alkylating agent chemotherapy exposure.

The impact of zinc and folic acid supplementation on sperm DNA methylation: results from the folic acid and zinc supplementation randomized clinical trial (FAZST).

OBJECTIVE: To determine if 6-month folic acid (5 mg) and zinc (30 mg) supplementation impacts sperm DNA methylation patterns. DESIGN: A multicenter, double-blind, block randomized, placebo-controlled trial titled "The Folic Acid and Zinc Supplementation Trial (FAZST)." SETTING: Infertility care centers. PATIENT(S): Male partners (18 years and older) from heterosexual couples (female partners aged 18-45 years) seeking fertility treatment were recruited. INTERVENTION(S): Men were randomized 1:1 to receive folic acid (5 mg) and elemental zinc (30 mg) (n = 713) or a matching placebo (n = 757) daily for 6 months. MAIN OUTCOME MEASURE(S): Sperm DNA methylation was analyzed using the EPIC methylation array (Illumina) at 6 months. Differential sperm DNA methylation was assessed at multiple levels (regional, single cytosine phosphate guanine, etc.). We additionally assessed the impact of supplementation on epigenetic age. RESULT(S): No significant differences were identified between the treatment and placebo groups although some trends appeared to be present. To determine if these trends were noteworthy, we implemented various permutations and found that the patterns we identified were no more than would be expected by random chance. CONCLUSION(S): The data presented here strongly suggest that this supplementation regimen is not effective at altering sperm DNA methylation. These data comport well with previous findings from the FAZST study that found no impact of supplementation on basic semen analysis parameters or live birth. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT01857310.

Epigenetic mechanisms within the sperm epigenome and their diagnostic potential.

The sperm epigenome contains a highly unique and specialized epigenetic landscape. Insightful questions need be asked about these epigenetic signatures and their predictive potential to assess the approximately 1 in 6 couples who experience infertility. Among those couples that do experience infertility, approximately half of the cases involve a male factor. Unfortunately, there is a significant lack of effective diagnostic tools in the male infertility space and thus clinicians are left with little data upon which they can formulate data driven treatment plans. Taking together this information and the striking prevalence of male infertility it's obvious that there is a need for improved diagnostic techniques for male infertility. Many studies have identified what appear to be clinically meaningful epigenetic alterations in sperm that may add utility in the diagnoses of infertility and improvement of pregnancy outcomes. Many researchers believe that continued analysis of these various epigenetic mechanisms may provide powerful predictive insight. In fact, there is promising current data suggesting that the predictive power of DNA methylation, Nuclear Proteins, and miRNA signatures in sperm likely can improve what is currently found with traditional diagnosis of male infertility. The focus of this review is to give a brief understanding to the field of epigenetics and the potential predictive power the sperm epigenome may hold in relation to improving the treatment and diagnosis of male infertility patients.

Harnessing the full potential of reproductive genetics and epigenetics for male infertility in the era of "big data".

The complexity of male reproductive impairment has hampered characterization of the underlying genetic causes of male infertility. However, in the last 20 years, more powerful and affordable tools to interrogate the genetic and epigenetic determinants of male infertility have accelerated the number of new discoveries in the characterization of male infertility. With this explosion of new data, integration in a systems-based approach-including complete phenotypic information-to male infertility is imperative. We briefly review the current understanding of genetic and epigenetic causes of male infertility and how findings may be translated into a practical component for the diagnosis and treatment of male infertility.

Towards a better testicular sperm extraction: novel sperm sorting technologies for non-motile sperm extracted by microdissection TESE.

Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It is characterized by a lack of spermatogenesis in the seminiferous tubules. Microdissection testicular sperm extraction (microTESE) has significantly improved testicular sperm retrieval rates compared to conventional techniques for NOA. Following testicular biopsy, the sperm is usually non-motile and contained within seminiferous tubules requiring extensive laboratory processing to find individual sperm sufficient for artificial reproductive technologies (ART). Current techniques include mechanical and enzymatic processing which is time-consuming and often damaging to sperm. We review novel techniques that may help improve sperm retrieval rates after microTESE including microfluidics (dielectrophoretic cell sorting, spiral channel sorting, and pinched flow fractionation), fluorescence-activated cell sorting (FACS), and magnetic-activated cell sorting (MACS).

Efficient assessment of efficacy in post-traumatic peripheral neuropathic pain patients: pregabalin in a randomized, placebo-controlled, crossover study.

BACKGROUND: Detecting the efficacy of novel analgesic agents in neuropathic pain is challenging. There is a critical need for study designs with the desirable characteristics of assay sensitivity, low placebo response, reliable pain recordings, low cost, short duration of exposure to test drug and placebo, and relevant and recruitable population. METHODS: We designed a proof-of-concept, double-blind, randomized, placebo-controlled, crossover study in patients with post-traumatic peripheral neuropathic pain (PTNP) to evaluate whether such a study design had the potential to detect efficacious agents. Pregabalin, known to be efficacious in neuropathic pain, was used as the active analgesic. We also assessed physical activity throughout the study. RESULTS: Twenty-five adults (20-70 years of age) with PTNP for ≥3 months entered a screening week and were then randomized to one of the two following treatment sequences: (1) pregabalin followed by placebo or (2) placebo followed by pregabalin. These 2-week treatment periods were separated by a 2-week washout period. Patients on pregabalin treatment received escalating doses to a final dosage of 300 mg/day (days 5-15). In an attempt to minimize placebo response, patients received placebo treatment during the screening week and the 2-week washout period. Average daily pain scores (primary endpoint) were significantly reduced for pregabalin versus placebo, with a mean treatment difference of -0.81 (95% confidence interval: -1.45 to -0.17; P = 0.015). CONCLUSION: The efficacy of pregabalin was similar to that identified in a large, parallel group trial in PTNP. Therefore, this efficient crossover study design has potential utility for future proof-of-concept studies in neuropathic pain.

Activity, pharmacokinetics and safety of lersivirine (UK-453,061), a next-generation nonnucleoside reverse transcriptase inhibitor, during 7-day monotherapy in HIV-1-infected patients.

OBJECTIVE: To investigate the effects on viral load and assess dose-response relationships, pharmacokinetics, safety and tolerability of lersivirine (UK-453,061), a next-generation nonnucleoside reverse transcriptase inhibitor, in asymptomatic HIV-1-infected patients. DESIGN: Randomized, double-blind, placebo-controlled, parallel group, multicenter phase IIa clinical study. METHODS: Forty-eight HIV-1-infected patients were enrolled for the study of once-daily or twice-daily lersivirine at total daily doses ranging from 20 to 1000 mg. The primary endpoint was the change in log10 plasma HIV-1 RNA viral load from baseline to day 8. Secondary endpoints related to pharmacokinetics, safety and tolerability and potential development of viral resistance and genotyping patterns. RESULTS: Patients treated with lersivirine achieved day 8 mean viral load reductions of 0.3, 0.8, 1.3 and 1.6 log10 after receiving 10, 30, 100 and 500 mg twice daily, respectively, and 0.9, 1.7 and 1.8 log10 after receiving 100, 500 and 750 mg once daily, respectively. Mean changes from baseline to day 8 were small in patients receiving placebo. For all dose regimens, plasma exposure increased approximately in line with lersivirine dose. Median plasma concentrations of lersivirine at steady state were above the IC90 for lersivirine at once-daily doses of at least 500 mg and twice-daily doses of at least 100 mg. The most commonly reported treatment-emergent adverse events were headache, fatigue and nausea. CONCLUSION: Seven-day monotherapy with lersivirine achieved mean viral load reductions up to 1.8 log10. Lersivirine was safe and well tolerated. Further studies of lersivirine in combination with other antiretroviral drugs to assess long-term durability of antiviral response, safety and tolerability are warranted.

Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir.

Antagonists of the human immunodeficiency virus type 1 (HIV-1) coreceptor, CCR5, are being developed as the first anti-HIV agents acting on a host cell target. We monitored the coreceptor tropism of circulating virus, screened at baseline for coreceptor tropism, in 64 HIV-1-infected patients who received maraviroc (MVC, UK-427,857) as monotherapy for 10 days. Sixty-two patients harbored CCR5-tropic virus at baseline and had a posttreatment phenotype result. Circulating virus remained CCR5 tropic in 60/62 patients, 51 of whom experienced an HIV RNA reduction from baseline of >1 log(10) copies/ml, indicating that CXCR4-using variants were not rapidly selected despite CCR5-specific drug pressure. In two patients, viral load declined during treatment and CXCR4-using virus was detected at day 11. No pretreatment factor predicted the emergence of CXCR4-tropic virus during maraviroc therapy in these two patients. Phylogenetic analysis of envelope (Env) clones from pre- and posttreatment time points indicated that the CXCR4-using variants probably emerged by outgrowth of a pretreatment CXCR4-using reservoir, rather than via coreceptor switch of a CCR5-tropic clone under selection pressure from maraviroc. Phylogenetic analysis was also performed on Env clones from a third patient harboring CXCR4-using virus prior to treatment. This patient was enrolled due to a sample labeling error. Although this patient experienced no overall reduction in viral load in response to treatment, the CCR5-tropic components of the circulating virus did appear to be suppressed while receiving maraviroc as monotherapy. Importantly, in all three patients, circulating virus reverted to predominantly CCR5 tropic following cessation of maraviroc.