MFN2 interacts with nuage-associated proteins and is essential for male germ cell development by controlling mRNA fate during spermatogenesis.

Mitochondria play a crucial role in spermatogenesis and are regulated by several mitochondrial fusion proteins. However, their functional importance associated with their structure formation and mRNA fate regulation during spermatogenesis remains unclear. Here, we show that mitofusin 2 (MFN2), a mitochondrial fusion protein, interacts with nuage-associated proteins (including MIWI, DDX4, TDRKH and GASZ) in mice. Conditional mutation of Mfn2 in postnatal germ cells results in male sterility due to germ cell developmental defects. Moreover, MFN2 interacts with MFN1, another mitochondrial fusion protein with a high-sequence similarity to MFN2, in testes to facilitate spermatogenesis. Simultaneous mutation of Mfn1 and Mfn2 in testes causes very severe infertile phenotypes. Importantly, we show that MFN2 is enriched in polysome fractions of testes and interacts with MSY2, a germ cell-specific DNA/RNA-binding protein, to control gamete-specific mRNA (such as Spata19) translational activity during spermatogenesis. Collectively, our findings demonstrate that MFN2 interacts with nuage-associated proteins and MSY2 to regulate male germ cell development by controlling several gamete-specific mRNA fates.

Disrupted intercellular bridges and spermatogenesis in fatty acyl-CoA reductase 1 knockout mice: A new model of ether lipid deficiency.

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation. The adult KO male mice had smaller testes and were infertile. H&E and immunofluorescent staining showed fewer germ cells in seminiferous tubules. Round spermatids were present but no elongated spermatids or spermatozoa were observed, suggesting a spermatogenesis arrest at this stage. Large multi-nucleated giant cells (MGC) were found lining the lumen of seminiferous tubules with many of them undergoing apoptosis. The immunofluorescent signal of TEX14, an essential component of intercellular bridges (ICB) between developing germ cells, was greatly reduced and mislocalized in KO testis, suggesting the disrupted ICBs as an underlying cause of MGC formation. Integrative analysis of our total testis RNA-sequencing results and published single-cell RNA-sequencing data unveiled cell type-specific molecular alterations underlying the spermatogenesis arrest. Many genes essential for late germ cell development showed dramatic downregulation, whereas genes essential for extracellular matrix dynamics and cell-cell interactions were among the most upregulated genes. Together, this work identified the cell type-specific requirement of ELs in spermatogenesis and suggested a critical role of Far1/ELs in the formation/maintenance of ICB during meiosis.

Effects of preconception lifestyle intervention in infertile women with obesity: The FIT-PLESE randomized controlled trial.

BACKGROUND: Women with obesity and infertility are counseled to lose weight prior to conception and infertility treatment to improve pregnancy rates and birth outcomes, although confirmatory evidence from randomized trials is lacking. We assessed whether a preconception intensive lifestyle intervention with acute weight loss is superior to a weight neutral intervention at achieving a healthy live birth. METHODS AND FINDINGS: In this open-label, randomized controlled study (FIT-PLESE), 379 women with obesity (BMI ≥ 30 kg/m2) and unexplained infertility were randomly assigned in a 1:1 ratio to 2 preconception lifestyle modification groups lasting 16 weeks, between July 2015 and July 2018 (final follow-up September 2019) followed by infertility therapy. The primary outcome was the healthy live birth (term infant of normal weight without major anomalies) incidence. This was conducted at 9 academic health centers across the United States. The intensive group underwent increased physical activity and weight loss (target 7%) through meal replacements and medication (Orlistat) compared to a standard group with increased physical activity alone without weight loss. This was followed by standardized empiric infertility treatment consisting of 3 cycles of ovarian stimulation/intrauterine insemination. Outcomes of any resulting pregnancy were tracked. Among 191 women randomized to standard lifestyle group, 40 dropped out of the study before conception; among 188 women randomized to intensive lifestyle group, 31 dropped out of the study before conception. All the randomized women were included in the intent-to-treat analysis for primary outcome of a healthy live birth. There were no significant differences in the incidence of healthy live births [standard 29/191(15.2%), intensive 23/188(12.2%), rate ratio 0.81 (0.48 to 1.34), P = 0.40]. Intensive had significant weight loss compared to standard (-6.6 ± 5.4% versus -0.3 ± 3.2%, P < 0.001). There were improvements in metabolic health, including a marked decrease in incidence of the metabolic syndrome (baseline to 16 weeks: standard: 53.6% to 49.4%, intensive 52.8% to 32.2%, P = 0.003). Gastrointestinal side effects were significantly more common in intensive. There was a higher, but nonsignificant, first trimester pregnancy loss in the intensive group (33.3% versus 23.7% in standard, 95% rate ratio 1.40, 95% confidence interval [CI]: 0.79 to 2.50). The main limitations of the study are the limited power of the study to detect rare complications and the design difficulty in finding an adequate time matched control intervention, as the standard exercise intervention may have potentially been helpful or harmful. CONCLUSIONS: A preconception intensive lifestyle intervention for weight loss did not improve fertility or birth outcomes compared to an exercise intervention without targeted weight loss. Improvement in metabolic health may not translate into improved female fecundity. TRIAL REGISTRATION: ClinicalTrials.gov NCT02432209.

RNA element discovery from germ cell to blastocyst.

Recent studies have shown that tissue-specific transcriptomes contain multiple types of RNAs that are transcribed from intronic and intergenic sequences. The current study presents a tool for the discovery of transcribed, unannotated sequence elements from RNA-seq libraries. This RNA Element (RE) discovery algorithm (REDa) was applied to a spectrum of tissues and cells representing germline, embryonic, and somatic tissues and examined as a function of differentiation through the first set of cell divisions of human development. This highlighted extensive transcription throughout the genome, yielding previously unidentified human spermatogenic RNAs. Both exonic and novel X-chromosome REs were subject to robust meiotic sex chromosome inactivation, although an extensive de-repression occurred in the post-meiotic stages of spermatogenesis. Surprisingly, 2.4% of the 10,395 X chromosome exonic REs were present in mature sperm. Transcribed genomic repetitive sequences, including simple centromeric repeats, HERVE and HSAT1, were also shown to be associated with RE expression during spermatogenesis. These results suggest that pervasive intergenic repetitive sequence expression during human spermatogenesis may play a role in regulating chromatin dynamics. Repetitive REs switching repeat classes during differentiation upon fertilization and embryonic genome activation was evident.

The relationship of plasma antioxidant levels to semen parameters: the Males, Antioxidants, and Infertility (MOXI) randomized clinical trial.

PURPOSE: The understanding of the role of plasma antioxidant levels in male fertility in the USA is limited. In a secondary analysis of the Males, Antioxidants, and Infertility (MOXI) randomized clinical trial, we sought to determine whether serum levels of vitamin E (α-tocopherol), zinc, and selenium were correlated with semen parameters and couple fertility outcomes. METHODS: This study is a secondary analysis of the MOXI clinical trial. The primary endpoints in this secondary analysis include semen parameters, and DNA fragmentation and clinical outcomes including pregnancy and live birth. Analyses were completed using Wilcoxon's rank-sum test and linear regression models. RESULTS: At baseline, the analysis included plasma labs for vitamin E (n = 131), selenium (n = 124), and zinc (n = 128). All baseline plasma values were in the normal ranges. There was no association between selenium, zinc, or vitamin E levels and semen parameters or DNA fragmentation. Baseline antioxidant levels in the male partners did not predict pregnancy or live birth among all couples. Among those randomized to placebo, baseline male antioxidant levels did not differ between those couples with live birth and those that did not conceive or have a live birth. CONCLUSIONS: Among men attending fertility centers in the USA, who have sufficient plasma antioxidant levels of zinc, selenium, or vitamin E, no association was observed between vitamins and semen parameters or clinical outcomes in couples with male infertility. Higher levels of antioxidants among men with circulating antioxidants in the normal range do not appear to confer benefit on semen parameters or male fertility.

Effect of an Active vs Expectant Management Strategy on Successful Resolution of Pregnancy Among Patients With a Persisting Pregnancy of Unknown Location: The ACT or NOT Randomized Clinical Trial.

Importance: Women with an early nonviable pregnancy of unknown location are at high risk of ectopic pregnancy and its inherent morbidity and mortality. Successful and timely resolution of the gestation, while minimizing unscheduled interventions, are important priorities. Objective: To determine if active management is more effective in achieving pregnancy resolution than expectant management and whether the use of empirical methotrexate is noninferior to uterine evacuation followed by methotrexate if needed. Design, Setting, and Participants: This multicenter randomized clinical trial recruited 255 hemodynamically stable women with a diagnosed persisting pregnancy of unknown location between July 25, 2014, and June 4, 2019, in 12 medical centers in the United States (final follow up, August 19, 2019). Interventions: Eligible patients were randomized in a 1:1:1 ratio to expectant management (n = 86), active management with uterine evacuation followed by methotrexate if needed (n = 87), or active management with empirical methotrexate using a 2-dose protocol (n = 82). Main Outcomes and Measures: The primary outcome was successful resolution of the pregnancy without change from initial strategy. The primary hypothesis tested for superiority of the active groups combined vs expectant management, and a secondary hypothesis tested for noninferiority of empirical methotrexate compared with uterine evacuation with methotrexate as needed using a noninferiority margin of -12%. Results: Among 255 patients who were randomized (median age, 31 years; interquartile range, 27-36 years), 253 (99.2%) completed the trial. Ninety-nine patients (39%) declined their randomized allocation (26.7% declined expectant management, 48.3% declined uterine evacuation, and 41.5% declined empirical methotrexate) and crossed over to a different group. Compared with patients randomized to receive expectant management (n = 86), women randomized to receive active management (n = 169) were significantly more likely to experience successful pregnancy resolution without change in their initial management strategy (51.5% vs 36.0%; difference, 15.4% [95% CI, 2.8% to 28.1%]; rate ratio, 1.43 [95% CI, 1.04 to 1.96]). Among active management strategies, empirical methotrexate was noninferior to uterine evacuation followed by methotrexate if needed with regard to successful pregnancy resolution without change in management strategy (54.9% vs 48.3%; difference, 6.6% [1-sided 97.5% CI, -8.4% to ∞]). The most common adverse event was vaginal bleeding for all of the 3 management groups (44.2%-52.9%). Conclusions and Relevance: Among patients with a persisting pregnancy of unknown location, patients randomized to receive active management, compared with those randomized to receive expectant management, more frequently achieved successful pregnancy resolution without change from the initial management strategy. The substantial crossover between groups should be considered when interpreting the results. Trial Registration: ClinicalTrials.gov Identifier: NCT02152696.

Abnormal fertility, acrosome formation, IFT20 expression and localization in conditional Gmap210 knockout mice.

GMAP210 (TRIP11) is a cis-Golgi network-associated protein and a Golgi membrane receptor for IFT20, an intraflagellar transport component essential for male fertility and spermiogenesis in mice. To investigate the role of GMAP210 in male fertility and spermatogenesis, floxed Gmap210 mice were bred with Stra8-iCre mice so that the Gmap210 gene is disrupted in spermatocytes and spermatids in this study. The Gmap210flox/flox: Stra8-iCre mutant mice showed no gross abnormalities and survived to adulthood. In adult males, testis and body weights showed no difference between controls and mutant mice. Low-magnification histological examination of the testes revealed normal seminiferous tubule structure, but sperm counts and fertility were significantly reduced in mutant mice compared with controls. Higher resolution examination of the mutant seminiferous epithelium showed that nearly all sperm had more oblong, abnormally shaped heads, while the sperm tails appeared to have normal morphology. Electron microscopy also revealed abnormally shaped sperm heads but normal axoneme core structure; some sperm showed membrane defects in the midpiece. In mutant mice, expression levels of IFT20 and other selective acrosomal proteins were significantly reduced, and their localization was also affected. Peanut-lectin, an acrosome maker, was almost absent in the spermatids and epididymal sperm. Mitochondrion staining was highly concentrated in the heads of sperm, suggesting that the midpieces were coiling around or aggregating near the heads. Defects in acrosome biogenesis were further confirmed by electron microscopy. Collectively, our findings suggest that GMAP210 is essential for acrosome biogenesis, normal mitochondrial sheath formation, and male fertility, and it determines expression levels and acrosomal localization of IFT20 and other acrosomal proteins.

Intrauterine insemination performance characteristics and post-processing total motile sperm count in relation to live birth for couples with unexplained infertility in a randomised, multicentre clinical trial.

STUDY QUESTION: Are intrauterine insemination (IUI) performance characteristics and post-processing total motile sperm count (TMC) related to live birth rate in couples with unexplained infertility? SUMMARY ANSWER: Patient discomfort with IUI and lower inseminate TMC were associated with a reduced live birth rate, while time from hCG injection to IUI, sperm preparation method and ultrasound guidance for IUI were not associated with live birth success. WHAT IS ALREADY KNOWN: We previously determined that some baseline characteristics of couples with unexplained infertility, including female age, duration of infertility, history of prior loss and income, were related to live birth rate across a course of ovarian stimulation and IUI treatment. However, the relationship between treatment outcomes and per-cycle characteristics, including ultrasound guidance for IUI, timing of IUI relative to hCG injection, difficult or painful IUI and inseminate TMC, are controversial, and most prior investigations have not evaluated live birth outcome. STUDY DESIGN, SIZE, DURATION: This was a secondary analyses of 2462 cycles from the Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) clinical trial. This prospective, randomised, multicentre clinical trial determined live birth rates following IUI after ovarian stimulation with clomiphene citrate, letrozole or gonadotropins in 854 couples with unexplained infertility. It was conducted between 2011 and 2014, and couples could undergo up to four consecutive treatment cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: AMIGOS was an NIH-sponsored Reproductive Medicine Network trial conducted at 12 clinical sites. Participants were women with unexplained infertility who were between 18 and 40 years of age. Cluster-weighted generalised estimating equations (GEE), which account for informative clustering of multiple IUI treatment cycles within the same patient, were used to determine associations between IUI performance characteristics, including inseminate TMC, and live birth rate. Efficiency curves were also generated to examine the relationship between inseminate TMC and live birth rate. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustment for treatment group and baseline factors previously associated with live birth across a course of OS-IUI treatment, patient discomfort during the IUI procedure was associated with a reduction in live birth rate (aRR 0.40 (0.16-0.96)). Time from hCG trigger injection to IUI was not significantly associated with outcome. Higher TMC was associated with greater live birth rate (TMC 15.1-20.0 million (14.8%) compared to ≤5 million (5.5%)) (aRR 2.09 (1.31-3.33)). However, live births did occur with TMC ≤ 1 million (5.1%). LIMITATIONS, REASONS FOR CAUTION: This investigation is a secondary analysis, and AMIGOS was not designed to address the present question. Since timed intercourse was allowed as part of the AMIGOS trial, we cannot rule out the possibility that any given pregnancy resulted from intercourse rather than IUI. WIDER IMPLICATIONS OF THE FINDINGS: Most factors associated with the performance of IUI were not significantly related to obtaining live birth. Our findings suggest that higher TMC inseminated leads to an increase in live birth rate up to TMC ~20 million. However, there may be no reasonable threshold below which live birth is not possible with IUI. STUDY FUNDING/COMPETING INTEREST(S): Funding was received through grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): U10 HD077680, U10 HD39005, U10 HD38992, U10 HD27049, U10 HD38998, U10 HD055942, HD055944, U10 HD055936 and U10 HD055925. This research was made possible by funding by the American Recovery and Reinvestment Act. Dr Hansen reports grants from NIH/NICHD and Yale University during the conduct of the study, grants from Roche Diagnostics and grants from Ferring International Pharmascience Center US outside the submitted work. Dr Peck reports support from Ferring Pharmaceuticals outside the submitted work. Dr Coward has nothing to disclose. Dr Wild reports grants from NICHD during the conduct of the study. Dr Trussell has nothing to disclose. Dr Krawetz reports grants from NICHD during the conduct of the study, grants from Merck and support from Taylor and Frances and from Springer, outside the submitted work. Dr Diamond reports grants from NIH/NICHD, Yale University, during the conduct of the study and support from Advanced Reproductive Care AbbVie, Bayer and ObsEva, outside the submitted work. Dr Legro reports support from Bayer, Kindex, Odega, Millendo and AbbVie and grants and support from Ferring, outside the submitted work. Dr Coutifaris reports grants from NICHD/NIH and personal fees from American Society for Reproductive Medicine, outside the submitted work. Dr Alvero has nothing to disclose. Dr Robinson reports grants from NIH during the conduct of the study. Dr Casson has nothing to disclose. Dr Christman reports grants from NICHD during the conduct of the study. Dr Santoro reports grants from NIH during the conduct of the study. Dr Zhang reports grants from NIH during the conduct of the study and support from Shangdong University outside the submitted work. TRIAL REGISTRATION NUMBER: n/a.

What human sperm RNA-Seq tells us about the microbiome.

PURPOSE: The study was designed to assess the capacity of human sperm RNA-seq data to gauge the diversity of the associated microbiome within the ejaculate. METHODS: Semen samples were collected, and semen parameters evaluated at time of collection. Sperm RNA was isolated and subjected to RNA-seq. Microbial composition was determined by aligning sequencing reads not mapped to the human genome to the NCBI RefSeq bacterial, viral and archaeal genomes following RNA-Seq. Analysis of microbial assignments utilized phyloseq and vegan. RESULTS: Microbial composition within each sample was characterized as a function of microbial associated RNAs. Bacteria known to be associated with the male reproductive tract were present at similar levels in all samples representing 11 genera from four phyla with one exception, an outlier. Shannon diversity index (p < 0.001) and beta diversity (unweighted UniFrac distances, p = 9.99e-4; beta dispersion, p = 0.006) indicated the outlier was significantly different from all other samples. The outlier sample exhibited a dramatic increase in Streptococcus. Multiple testing indicated two operational taxonomic units, S. agalactiae and S. dysgalactiae (p = 0.009), were present. CONCLUSION: These results provide a first look at the microbiome as a component of human sperm RNA sequencing that has sufficient sensitivity to identify contamination or potential pathogenic bacterial colonization at least among the known contributors.

Fertility Related Quality of Life, Gonadal Function and Erectile Dysfunction in Male Partners of Couples with Unexplained Infertility.

PURPOSE: We sought to determine whether lower fertility related quality of life or depression in men of couples with unexplained infertility is associated with low total testosterone levels, abnormal semen quality or erectile dysfunction. MATERIALS AND METHODS: This study is a secondary analysis of a large, multicenter, randomized controlled trial in couples with unexplained infertility. Male partners underwent baseline semen analysis with measurement of fasting total testosterone and gonadotropin. They also completed surveys, including the FertiQOL (Fertility Quality of Life), the PHQ-9 (Patient Health Questionnaire-9) and the IIEF (International Index of Erectile Function). The primary study outcomes were total testosterone with low total testosterone defined as less than 264 ng/dl, semen parameters and the IIEF score. We performed multivariable logistic regression analyses adjusted for patient age, race, body mass index, education, smoking, alcohol use, infertility duration and comorbidity. RESULTS: A total of 708 men with a mean ± SD age of 34.2 ± 5.6 were included in study. Of the men 59 (8.3%) had a PHQ-9 score of 5 or greater, which was consistent with depression, 99 (14.0%) had low total testosterone and 63 (9.0%) had mild or worse erectile dysfunction. Neither the FertiQOL score nor depression was associated with total testosterone or any semen parameter. The FertiQOL score was inversely associated with erectile dysfunction (for every 5-point score decline AOR 1.30, 95% CI 1.16-1.46). Depressed men were significantly more likely to have erectile dysfunction than nondepressed men (AOR 6.31, 95% CI 3.12-12.77). CONCLUSIONS: In men in couples with unexplained infertility lower fertility related quality of life and depression are strongly associated with erectile dysfunction. However, neither is associated with spermatogenesis or testosterone levels. Erectile dysfunction in infertile men merits longitudinal investigation in future studies.

Immune regulatory molecules as modifiers of semen and fertility: A review.

Declining fertility rates in both human and animals is a cause for concern. While many of the infertility cases are due to known causes, idiopathic infertility is reported in 30% of the infertile couples. In such cases, 18% of the infertile males carry antisperm antibodies (ASAs). Such data are lacking in livestock, wherein 20-30% of the animals are being culled due to low fertility. In males, the blood-testis barrier (BTB) and biomolecules in the semen provide an immuno-tolerant microenvironment for spermatozoa as they traverse the immunologic milieu of both the male and female reproductive tracts. For example, insults from environmental contaminants, infections and inflammatory conditions are likely to impact the immune privilege state of the testis and fertility. The female mucosal immune system can recognize allogenic spermatozoa-specific proteins affecting sperm kinematics and sperm-zona binding leading to immune infertility. Elucidating the functions and pathways of the immune regulatory molecules associated with fertilization are prerequisites for understanding their impact on fertility. An insight into biomolecules associated with spermatozoal immune tolerance may generate inputs to develop diagnostic tools and modulate fertility. High-throughput sequencing technologies coupled with bioinformatics analyses provides a path forward to define the array of molecules influencing pregnancy outcome. This review discusses the seminal immune regulatory molecules from their origin in the testis until they traverse the uterine environment enabling fertilization and embryonic development. Well-designed experiments and the identification of biomarkers may provide a pathway to understand the finer details of reproductive immunology that will afford personalized therapies.

The efficiency of single institutional review board review in National Institute of Child Health and Human Development Cooperative Reproductive Medicine Network-initiated clinical trials.

BACKGROUND/AIMS: Timely review of research protocols by institutional review boards leads to more rapid initiation of clinical trials, which is critical to expeditious translation from bench to bedside. This observational study examined the impact of a single institutional review board on time and efforts required to initiate clinical trials by the National Institute of Child Health and Human Development Cooperative Reproductive Medicine Network. METHODS: Collection of data from the same six main clinical sites for three current clinical trials and two past clinical trials, including time from institutional review board submission to approval, pages submitted, consent form length, number of required attachments, other regulatory requirements, order of review at central or local sites, and language in documents at individual participating sites. Results from two past clinical trials were also included. RESULTS: While time required for actual institutional review board submission's review and initial approval was reduced with use of a single institutional review board for multicenter trials (from a mean of 66.7-24.0 days), total time was increased (to a mean of 111.2 or 123.3 days). In addition to single institutional review board approval, all institutions required local approval of some components (commonly consent language and use of local language), which varied considerably. The single institutional review board relied on local institutions for adding or removing personnel, conflict of interest review, and auditing of activities. CONCLUSION: A single institutional review board reduced time for initial review and approval of protocols and informed consents, although time for the entire process was increased, as individual institutions retained oversight of components of required regulatory review. In order to best achieve the National Institute of Health's goals for improved efficiency in initiation and conduct of multisite clinical research, greater coordination with local institutional review boards is key to streamlining and accelerating initiation of multisite clinical research.

Letrozole, Gonadotropin, or Clomiphene for Unexplained Infertility.

BACKGROUND: The standard therapy for women with unexplained infertility is gonadotropin or clomiphene citrate. Ovarian stimulation with letrozole has been proposed to reduce multiple gestations while maintaining live birth rates. METHODS: We enrolled couples with unexplained infertility in a multicenter, randomized trial. Ovulatory women 18 to 40 years of age with at least one patent fallopian tube were randomly assigned to ovarian stimulation (up to four cycles) with gonadotropin (301 women), clomiphene (300), or letrozole (299). The primary outcome was the rate of multiple gestations among women with clinical pregnancies. RESULTS: After treatment with gonadotropin, clomiphene, or letrozole, clinical pregnancies occurred in 35.5%, 28.3%, and 22.4% of cycles, and live birth in 32.2%, 23.3%, and 18.7%, respectively; pregnancy rates with letrozole were significantly lower than the rates with standard therapy (gonadotropin or clomiphene) (P=0.003) or gonadotropin alone (P<0.001) but not with clomiphene alone (P=0.10). Among ongoing pregnancies with fetal heart activity, the multiple gestation rate with letrozole (9 of 67 pregnancies, 13%) did not differ significantly from the rate with gonadotropin or clomiphene (42 of 192, 22%; P=0.15) or clomiphene alone (8 of 85, 9%; P=0.44) but was lower than the rate with gonadotropin alone (34 of 107, 32%; P=0.006). All multiple gestations in the clomiphene and letrozole groups were twins, whereas gonadotropin treatment resulted in 24 twin and 10 triplet gestations. There were no significant differences among groups in the frequencies of congenital anomalies or major fetal and neonatal complications. CONCLUSIONS: In women with unexplained infertility, ovarian stimulation with letrozole resulted in a significantly lower frequency of multiple gestation but also a lower frequency of live birth, as compared with gonadotropin but not as compared with clomiphene. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01044862.).

A history of why fathers' RNA matters.

Having been debated for many years, the presence and role of spermatozoal RNAs is resolving, and their contribution to development is now appreciated. Data from different species continue show that sperm contain a complex suite of coding and noncoding RNAs that play a role in an individual's life course. Mature sperm RNAs provide a retrospective of spermatogenesis, with their presence and abundance reflecting sperm maturation, fertility potential, and the paternal contribution to the developmental path the offspring may follow.Sperm RNAs delivered upon fertilization provide some of the initial contacts with the oocyte, directly confront the maternal with the paternal contribution as a prelude to genome consolidation. Following syngamy, early embryo development may in part be modulated by paternal RNAs that can include epidydimal passengers. This provides a direct path to relay an experience and then initiate a paternal response to the environment to the oocyte and beyond. Their epigenetic impact is likely felt prior to embryonic genome activation when the population of sperm delivered transcripts markedly changes. Here, we review the insights gained from sperm RNAs over the years, the subtypes, and the caveats of the RNAs described. We discuss the role of sperm RNAs in fertilization and embryo development, and their possible mechanism(s) influencing offspring phenotype. Approaches to meet the future challenges as the study of sperm RNAs continues, include, elucidating the potential mechanisms underlying how paternal allostatic load, the constant adaptation of health to external conditions, may be relayed by sperm RNAs to affect future generations.

A crossover-crossback prospective study of dibutyl-phthalate exposure from mesalamine medications and serum reproductive hormones in men.

BACKGROUND: Phthalates, such as dibutyl phthalate (DBP), are endocrine disruptors used in some medication coatings e.g., mesalamine to treat inflammatory bowel disease (IBD). OBJECTIVES: Taking advantage of different mesalamine formulations with/without DBP, we assessed whether DBP from mesalamine (>1000x background) altered serum hormones. METHODS: Men (N=73) with IBD participated in a crossover-crossback prospective study and provided up to 6 serum samples (2:baseline, 2:crossover, 2:crossback). Men on non-DBP mesalamine (background) at baseline crossed-over for 4 months to DBP-mesalamine (high) and then crossed-back for 4 months to non-DBP mesalamine (B1HB2-arm) and vice versa for men on DBP-mesalamine at baseline (H1BH2-arm). We divided H1BH2-arm at the median (H1<3yrs or H1≥3yrs). We estimated crossover and crossback % changes in serum reproductive hormones using multivariable linear mixed effect models. RESULTS: When B1HB2-arm (26 men,134 samples) crossed-over, luteinizing hormone decreased 13.9% (95% confidence interval(CI): -23.6,-3.0) and testosterone, inhibin-B, and follicle-stimulating hormone (FSH) marginally decreased; after crossback all increased 8-14%. H1BH2-arm, H1≥3yrs (25 men,107samples) had no changes at crossover or crossback whereas in H1BH2-arm,H1<3yrs (22 men,100 samples) after crossover, inhibin-B increased 13.2% (CI: 4.2,22.9), FSH decreased 9.9% (CI: -17.9,-1.1) and after crossback, inhibin-B further increased 11.3%, and FSH marginally increased. CONCLUSIONS: High-DBP exposure may disrupt pituitary-gonadal hormones that largely reversed after exposure removal, but only in men with no or short previous high-exposure history. Paradoxically, men with longer duration of high-DBP exposure, exposure removal did not change hormone levels, suggesting that long-term high-DBP exposure may alter the pituitary-gonadal axis and make it insensitive to exposure changes.

Letrozole versus clomiphene for infertility in the polycystic ovary syndrome.

BACKGROUND: Clomiphene is the current first-line infertility treatment in women with the polycystic ovary syndrome, but aromatase inhibitors, including letrozole, might result in better pregnancy outcomes. METHODS: In this double-blind, multicenter trial, we randomly assigned 750 women, in a 1:1 ratio, to receive letrozole or clomiphene for up to five treatment cycles, with visits to determine ovulation and pregnancy, followed by tracking of pregnancies. The polycystic ovary syndrome was defined according to modified Rotterdam criteria (anovulation with either hyperandrogenism or polycystic ovaries). Participants were 18 to 40 years of age, had at least one patent fallopian tube and a normal uterine cavity, and had a male partner with a sperm concentration of at least 14 million per milliliter; the women and their partners agreed to have regular intercourse with the intent of conception during the study. The primary outcome was live birth during the treatment period. RESULTS: Women who received letrozole had more cumulative live births than those who received clomiphene (103 of 374 [27.5%] vs. 72 of 376 [19.1%], P=0.007; rate ratio for live birth, 1.44; 95% confidence interval, 1.10 to 1.87) without significant differences in overall congenital anomalies, though there were four major congenital anomalies in the letrozole group versus one in the clomiphene group (P=0.65). The cumulative ovulation rate was higher with letrozole than with clomiphene (834 of 1352 treatment cycles [61.7%] vs. 688 of 1425 treatment cycles [48.3%], P<0.001). There were no significant between-group differences in pregnancy loss (49 of 154 pregnancies in the letrozole group [31.8%] and 30 of 103 pregnancies in the clomiphene group [29.1%]) or twin pregnancy (3.4% and 7.4%, respectively). Clomiphene was associated with a higher incidence of hot flushes, and letrozole was associated with higher incidences of fatigue and dizziness. Rates of other adverse events were similar in the two treatment groups. CONCLUSIONS: As compared with clomiphene, letrozole was associated with higher live-birth and ovulation rates among infertile women with the polycystic ovary syndrome. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00719186.).

Preconception urinary phthalate concentrations and sperm DNA methylation profiles among men undergoing IVF treatment: a cross-sectional study.

STUDY QUESTION: Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF? SUMMARY ANSWER: Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure. WHAT IS KNOWN ALREADY: Several phthalate compounds and their metabolites are known endocrine disrupting compounds and are pervasive environmental contaminants. Rodent studies report that prenatal phthalate exposures induce sperm DMRs, but the influence of preconception phthalate exposure on sperm DNA methylation in humans is unknown. STUDY DESIGN, SIZE, DURATION: An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS). PARTICIPANTS/MATERIALS, SETTING, METHODS: The first 48 couples provided a spot urine sample on the same day as semen sample procurement. Sperm DNA methylation was assessed with the HumanMethylation 450 K array. Seventeen urinary phthalate and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) metabolite concentrations were measured from spot urine samples. The A-clust algorithm was employed to identify co-regulated regions. DMRs associated with urinary metabolite concentrations were identified via linear models, corrected for false discovery rate (FDR). MAIN RESULTS AND ROLE OF CHANCE: Adjusting for age, BMI, and current smoking, 131 DMRs were associated with at least one urinary metabolite. Most sperm DMRs were associated with anti-androgenic metabolites, including mono(2-ethylhexyl) phthalate (MEHP, n = 83), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP, n = 16), mono-n-butyl phthalate (MBP, n = 22) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl (MCOCH, n = 7). The DMRs were enriched in lincRNAs as well as in regions near coding regions. Functional analyses of DMRs revealed enrichment of genes related to growth and development as well as cellular function and maintenance. Finally, 13% of sperm DMRs were inversely associated with high quality blastocyst-stage embryos after IVF. LIMITATIONS, REASONS FOR CAUTION: Our modest sample size only included 48 males and additional larger studies are necessary to confirm our observed results. Non-differential misclassification of exposure is also a concern given the single spot urine collection. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study to report that preconception urinary phthalate metabolite concentrations are associated with sperm DNA methylation in humans. These results suggest that paternal adult environmental conditions may influence epigenetic reprogramming during spermatogenesis, and in turn, influence early-life development. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests.

Sexual function in infertile women with polycystic ovary syndrome and unexplained infertility.

BACKGROUND: While female sexual dysfunction is a frequent occurrence, characteristics in infertile women are not well delineated. Furthermore, the impact of infertility etiology on the characteristics in women with differing androgen levels observed in women with polycystic ovary syndrome and unexplained infertility has not been assessed. OBJECTIVE: The objective of the study was to determine the characteristics of sexual dysfunction in women with polycystic ovary syndrome and unexplained infertility. STUDY DESIGN: A secondary data analysis was performed on 2 of Eunice Kennedy Shriver National Institute of Child Health and Human Development Cooperative Reproductive Medicine Networks clinical trials: Pregnancy in Polycystic Ovary Syndrome Study II and Assessment of Multiple Intrauterine Gestations From Ovarian Stimulation. Both protocols assessed female sexual function using the Female Sexual Function Inventory and the Female Sexual Distress Scale. RESULTS: Women with polycystic ovary syndrome had higher weight and body mass index than women with unexplained infertility (each P < .001), greater phenotypic (Ferriman-Gallwey hirsutism score, sebum score, and acne score; each P < .001), and hormonal (testosterone, free testosterone, and dehydroepiandrosterone; each P < .001) evidence of androgen excess. Sexual function scores, as assessed by the Female Sexual Function Inventory, were nearly identical. The Female Sexual Distress Scale total score was higher in women with polycystic ovary syndrome. The mean Female Sexual Function Inventory total score increased slightly as the free androgen index increased, mainly as a result of the desire subscore. This association was more pronounced in the women with unexplained infertility. CONCLUSION: Reproductive-age women with infertility associated with polycystic ovary syndrome and unexplained infertility, despite phenotypic and biochemical differences in androgenic manifestations, do not manifest clinically significant differences in sexual function.

Chromatin and extracellular vesicle associated sperm RNAs.

A diverse pool of RNAs remain encapsulated within the transcriptionally silent spermatozoon despite the dramatic reduction in cellular and nuclear volume following cytoplasm/nucleoplasm expulsion. The impact of this pronounced restructuring on the distribution of transcripts inside the sperm essentially remains unknown. To define their compartmentalization, total RNA >100 nt was extracted from sonicated (SS) mouse spermatozoa and detergent demembranated sucrose gradient fractionated (Cs/Tx) sperm heads. Sperm RNAs predominately localized toward the periphery. The corresponding distribution of transcripts and thus localization and complexity were then inferred by RNA-seq. Interestingly, the number of annotated RNAs in the CsTx sperm heads exhibiting reduced peripheral enrichment was restricted. However this included Cabyr, the calcium-binding tyrosine phosphorylation-regulated protein encoded transcript. It is present in murine zygotes prior to the maternal to the zygotic transition yet absent in oocytes, consistent with the delivery of internally positioned sperm-borne RNAs to the embryo. In comparison, transcripts enriched in sonicated sperm contributed to the mitochondria and exosomes along with several nuclear transcripts including the metastasis associated lung adenocarcinoma transcript 1 (Malat1) and several small nucleolar RNAs. Their preferential peripheral localization suggests that chromatin remodeling during spermiogenesis is not limited to nucleoproteins as part of the nucleoprotein exchange.

The protein and transcript profiles of human semen.

The increasing use of "-omics" (genomic, transcriptomic, proteomic, epigenomic, and metabolomic) high-throughput measurement technologies over the past decade is beginning to reveal the complexity of human biology and physiology through the interactions of DNA, RNA, related proteins and small molecules. In reproductive medicine, the majority of this work, has thus far focused on the female factors, e.g., the oocyte, since they provide both the environment and the majority of elements required for embryogenesis. State-of-the-art sequencing and computational analyses have enabled a deeper understanding of the underlying components. Contrary to being simply a silent delivery vehicle to the oocyte of the packaged male DNA, sperm provide both a specific epigenetically marked genome together with a complex population of RNAs and proteins that are crucial for early embryogenesis. In addition to the sperm, seminal fluid appears to serve multiple roles providing a supplementary series of components that allow the sperm to successfully reach and fertilize the oocyte and prepare the female immune system to tolerate the semiallosteric embryo. A global analysis and review of what is presently known regarding the unique role of each component of the male factor and their associated interactions begins to shed light on this emergent field.