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Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Albumin, a vital protein in cell culture systems, is derived from whole blood or blood products. The culture of human gametes and developing embryos for assisted reproductive technology (ART) uses albumin of human origin. Human serum albumin (HSA) is derived from expired blood obtained from blood banks. This blood has been stored in polyvinyl chloride bags made clear and flexible with di-2-ethylhexyl phthalate (DEHP). However, DEHP can leach from the bags into stored blood and cofractionate with HSA during albumin isolation. DEHP and its metabolite, mono-ethylhexyl phthalate, are known endocrine disruptors that are reported to have negative effects when directly supplemented in media for in vitro fertilization using gametes from a variety of animals. Therefore, the contamination of ART media with DEHP and mono-ethylhexyl phthalate through HSA supplementation may affect the outcomes of ART procedures. Although the embryology laboratory is strictly monitored to prevent a wide variety of contaminations, phthalate contamination of HSA has not been broadly examined. This review outlines the function of HSA in ART procedures and the production of HSA from whole blood. Finally, the review highlights the effects of acute phthalate exposures on gametes during in vitro procedures. Phthalates found in human serum albumin are present in media used for ART at levels that impair developmental endpoints in model species.

OBJECTIVE: To examine the association between cultivable vaginal Lactobacillus and fecundability in Kenyan women attempting non-medically assisted conception DESIGN: Prospective preconception cohort SETTING: Nairobi and Mombasa, Kenya PATIENTS: Women trying to conceive who reported ≤3 months of pre-enrollment conception attempt time EXPOSURE: Cultivable Lactobacillus (primary), Lactobacillus morphotypes on Gram stain (secondary) MAIN OUTCOME MEASURES: Participants reported first day of last menstrual period and recent sexual behavior, underwent pregnancy testing, and provided vaginal specimen samples for Lactobacillus culture and Gram stain at ≤6 monthly preconception visits. The outcome was fecundability – the per-menstrual cycle probability of pregnancy. Associations between 1) cultivable Lactobacillus and 2) Lactobacillus morphotypes on Gram stain at the visit prior to each pregnancy test and fecundability were estimated using proportional probabilities models to generate fecundability ratios (FRs). RESULTS: 458 women contributed 1,376 menstrual cycles. At enrollment, 65.3% (n=299) of participants had cultivable Lactobacillus, 47.4% (n=217) had cultivable hydrogen peroxide producing Lactobacillus, and 64.6% (n=296) had Lactobacillus detected on Gram stain. In unadjusted analysis, there was no association between cultivable Lactobacillus at the visit prior and fecundability (FR 0.92, 95%CI 0.73–1.16); results were similar after adjustment for age, frequency of condomless sex, and study site (adjusted FR 0.92, 95%CI 0.72–1.18). Lactobacillus on Gram stain at the visit prior was associated with modestly higher fecundability (adjusted FR 1.18, 95%CI 0.92–1.51). CONCLUSIONS: Cultivable Lactobacillus was not associated with fecundability while Lactobacillus morphotypes detected on Gram stain were somewhat associated with increased fecundability. The relationship between vaginal Lactobacillus and fecundity may be species-specific.

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs)., Male germ cell production is a metabolically-driven and apoptosis-prone process. This study shows that MLX, a nutrient-sensing transcription factor and member of the MYC network, is not required for embryonic development but is necessary for male murine fertility and male germ cell tumor survival; loss of MLX results in altered metabolism, activation of stress pathways and germ cell apoptosis in the testes.

STUDY QUESTION Is bacterial vaginosis (BV) associated with fecundability? SUMMARY ANSWER Women with BV may be at increased risk for sub-fecundity. WHAT IS KNOWN ALREADY While BV has been associated with poor IVF outcomes, the association between vaginal microbiota disruption and non-medically assisted conception has not been thoroughly explored. STUDY DESIGN, SIZE, DURATION Kenyan women with fertility intent were enrolled in prospective cohort that included monthly preconception visits with vaginal fluid specimen collection and pregnancy testing. Four hundred fifty-eight women attempting pregnancy for ≤3 menstrual cycles at enrollment were eligible for this fecundability analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS At monthly preconception visits, participants reported the first day of last menstrual period and sexual behavior, underwent pregnancy testing and provided vaginal specimens. Discrete time proportional probabilities models were used to estimate fecundability ratios (FRs) and 95% CI in menstrual cycles with and without BV (Nugent score ≥ 7) at the visit prior to each pregnancy test. We also assessed the association between persistent BV (BV at two consecutive visits) and fecundability. MAIN RESULTS AND THE ROLE OF CHANCE Participants contributed 1376 menstrual cycles; 18.5% (n = 255) resulted in pregnancy. After adjusting for age, frequency of condomless sex and study site, BV at the visit prior to pregnancy testing was associated with a 17% lower fecundability (adjusted FR (aFR) 0.83, 95% CI 0.6–1.1). Persistent BV was associated with a 43% reduction in fecundability compared to cycles characterized by optimal vaginal health (aFR 0.57, 95% CI 0.4–0.8). LIMITATIONS, REASONS FOR CAUTION Detection of vaginal microbiota disruption using Gram stain and a point-of-care test for elevated sialidase identified a non-optimal vaginal environment, but these non-specific methods may miss important relationships that could be identified by characterizing individual vaginal bacteria and bacterial communities using molecular methods. In addition, results may be subject to residual confounding by condomless sex as this was reported for the prior month rather than for the fertile window during each cycle. WIDER IMPLICATIONS OF THE FINDINGS Given the high global prevalence of BV and infertility, an association between BV and reduced fecundability could have important implications for a large number of women who wish to conceive. Multi-omics approaches to studying the vaginal microbiota may provide key insights into this association and identify potential targets for intervention. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a National Institutes of Health grant (NICHD R01 HD087346-R.S.M.). R.S.M. received additional support for mentoring (NICHD K24 HD88229). E.M.L. was supported by pre- and post-doctoral fellowships (NIAID T32 AI07140, NICHD F32 HD100202). Data collection and management were made possible using REDCap electronic data capture tools hosted at the University of Washington’s Institute of Translational Health Science supported by grants from NCATS/NIH (UL1 TR002319). The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. R.S.M. receives research funding, paid to the University of Washington, from Hologic Corporation, and has received honoraria for consulting from Lupin Pharmaceuticals. L.E.M. receives research funding, paid to the University of Washington, from Hologic Corporation, and has received honoraria for service on scientific advisory boards from Hologic and Nabriva Therapeutics. TRIAL REGISTRATION NUMBER N/A.

Male germ cell production is a metabolically-driven and apoptosis-prone process. Here we show that the glucose-sensing transcription factor MLX, and its binding partner MondoA, are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss ofMlxresults in altered metabolism as well as activation of multiple stress pathways and germ cell apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific germ cell transcripts and proteins, manifesting as oligoasthenoteratozoospermia (OAT). Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, apoptotic effectors and obligate components of male-specific germ cell development. Thesein vivoandin vitrostudies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors.Highlights-The MAX-like bHLHLZ protein MLX is required for male fertility, but not embryonic development.-MLX and its heterodimeric partner MondoA are each required for both male fertility and survival of male germ cell tumors.-Genomic analysis identifies direct MLX targets associated with metabolism, stress and male germ cell development.-Loss of MLX alters MYC network genome occupancy and transcriptional output.

Once unimaginable, fertility management is now a nationally established part of cancer care in institutions, from academic centers to community hospitals to private practices. Over the last two decades, advances in medicine and reproductive science have made it possible for men, women and children to be connected with an oncofertility specialist or offered fertility preservation soon after a cancer diagnosis. The Oncofertility Consortium's National Physicians Cooperative is a large-scale effort to engage physicians across disciplines – oncology, urology, obstetrics and gynecology, reproductive endocrinology, and behavioral health – in clinical and research activities to enable significant progress in providing fertility preservation options to children and adults. Here, we review the structure and function of the National Physicians Cooperative and identify next steps.

Histone variants expand chromatin functions in eukaryote genomes. H2A.B genes are testis-expressed short histone H2A variants that arose in placental mammals. Their biological functions remain largely unknown. To investigate their function, we generated a knockout (KO) model that disrupts all 3 H2A.B genes in mice. We show that H2A.B KO males have globally altered chromatin structure in postmeiotic germ cells. Yet, they do not show impaired spermatogenesis or testis function. Instead, we find that H2A.B plays a crucial role postfertilization. Crosses between H2A.B KO males and females yield embryos with lower viability and reduced size. Using a series of genetic crosses that separate parental and zygotic contributions, we show that the H2A.B status of both the father and mother, but not of the zygote, affects embryonic viability and growth during gestation. We conclude that H2A.B is a novel parental-effect gene, establishing a role for short H2A histone variants in mammalian development. We posit that parental antagonism over embryonic growth drove the origin and ongoing diversification of short histone H2A variants in placental mammals., The unusual short histone variant H2A.B is a novel parental-effect gene that plays an important role in early mammalian development. Parental antagonism over embryonic growth resource allocation may have driven the origin and ongoing diversification of short histone H2A variants in placental mammals.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

Globally, ∼1 in 15 men of reproductive age are infertile, yet the precise mechanisms underlying their gamete failure are unknown. Although a semen analysis is performed to determine fertilizing potential, the diagnostic suitability of this analysis has been questioned in several reports, as many men, classified as infertile according to their semen analysis, subsequently turn out to be fertile. Herein, we have used a quantitative (phospho)-proteomic analysis, using enrichment on titanium dioxide followed by ion-trap mass spectrometry (LC-MS/MS), to compare the semen of infertile versus fertile males. One protein, namely outer dense fiber 1 (ODF1), was dramatically reduced in infertile males. Using specific antibodies, we then screened the gametes of a cohort of suspected infertile men and demonstrated a reduction in the amount of ODF1 compared with fertile controls. Stress treatment of sperm deficient in ODF1 caused the head to decapitate, suggesting why these gametes fail to initiate fertilization. Interestingly, electron micrographs of ODF1-deficient spermatozoa revealed an abnormal connecting piece, indicating several developmental defects with both the implantation plate and the thin laminated fibers. In some cases, the implantation plate appeared to be reduced in size or was overburdened by granular material near the connecting piece. Hence, a strong reduction ODF1 is a marker of idiopathic male infertility and a potential driver of this condition.

Aims: To assess if marijuana consumption – prevalent among men of reproductive age and becoming widespread due to decriminalization – is associated with changes in semen parameters. Marijuana’s active metabolite, tetrahydrocannabinol, can alter signaling pathways within spermatozoa, affecting spermatogenesis and fertility. Methods: We prospectively evaluated semen analyses (SA) from men presenting for infertility evaluation at one institution from July 2017 to April 2018. Participants completed a reproductive health questionnaire including items regarding marijuana consumption. SA was performed in accordance with World Health Organization (WHO) 5th Edition criteria. SA parameters included volume (ml), concentration (million/ml), motility (%), progressive motility (%), and Tygerberg strict morphology (%). Results: A total of 409 patients completed the questionnaire; 174 (43%) men reported marijuana use (ever-users). Current and past users comprised 71 (17%) and 103 (25%), respectively. Compared with never-users, current and past users had a significantly higher likelihood of abnormal sperm strict morphology (33.1% versus 50.7% and 53.4%, respectively; p Conclusion: Marijuana use is common among men presenting for fertility evaluation, and may have a detrimental effect on semen quality, particularly morphology and volume, but may be protective against abnormal sperm motility. Large, prospective studies of both semen quality and fertility in this growing, at-risk population are warranted.

Supplemental Digital Content is Available in the Text., Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission.

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of FSHβ. We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSHβ consisting of amino acids 33 to 53 (FSHβ (33–53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33–53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33–53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33–53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33–53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHβ (33–53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.

Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.

Objectives: Genitourinary tract samples are required to investigate male HIV-1 infectivity. Because semen collection is often impractical, the acceptability, feasibility and validity of post-prostatic massage fluid/ urine (post-PMF/U) was evaluated for studying male genitourinary HIV-1 shedding. Methods: HIV-1-seropositive men were evaluated after 48 h of sexual abstinence. At each visit, a clinician performed prostatic massage, then post-PMF/U and blood were collected. Participants provided semen specimens 1 week later. An audio computer-assisted self-interview (ACASI) administered after each specimen collection evaluated acceptability, adherence to instructions and recent genitourinary symptoms. HIV-1 RNA was quantified using a real-time PCR assay. Detection and quantitation of HIV-1 RNA and stability over visits were compared for semen, post-PMF/U and blood. Results: Post-PMF/U was successfully obtained at 106 visits (64%) and semen at 136 visits (81%, p

Intratesticular retinoic acid is necessary for spermatogenesis, but the relationship between intratesticular retinoic acid and sperm quality in man has not been studied. We hypothesized that intratesticular concentrations of retinoic acid would be lower in men with abnormal semen analyses compared to men with normal semen analyses. We recruited men requiring scrotal or penile surgery in a pilot observational study examining the relationship between sperm quality and intratesticular and serum retinoic acid. Twenty-four men provided two pre-operative blood and semen samples, and underwent a testicular biopsy during surgery. Serum and tissue all-trans and 13-cis retinoic acid and reproductive hormones were measured by LC/MS/MS and radioimmunoassays, respectively. Seven men had abnormal semen analyses by at least one WHO criteria and 17 men were normal. In men with abnormal semen, the median (25th, 75th percentile) intratesticular 13-cis retinoic acid was 0.14 (0.08, 0.25) pmol/gram tissue compared with 0.26 (0.18, 0.38) pmol/gram tissue in men with normal semen (p = 0.04). There were no significant differences in intratesticular all-trans retinoic acid or serum reproductive hormones between men with normal and abnormal semen analyses. Intratesticular 13-cis retinoic acid is significantly lower in men with abnormal semen analyses compared to men with normal semen analyses. Lower intratesticular 13-cis retinoic acid concentrations may be due to decreased biosynthesis or increased metabolism in the testes. Further investigation of the relationship between intratesticular 13-cis retinoic acid and poor sperm quality is warranted to determine if this association is present in infertile men.

BACKGROUND AND PURPOSE Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB1 cannabinoid receptor agonist, and Δ9-tetrahydracannabinol (Δ9-THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of Δ9-THC on male fecundity were determined. EXPERIMENTAL APPROACH Effects of Δ9-THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of Δ9-THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by Δ9-THC-treated males. KEY RESULTS Pretreatment of sperm for 15 min with 1 µM Δ9-THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 µM WIN 55,212-2 or 10 µM Δ9-THC for 30 min reduced sperm ATP levels. In sperm lacking CB1 receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of Δ9-THC persisted. Administration of 50 mg·kg−1Δ9-THC to male mice just before mating caused a 20% decrease in embryonic litter size. CONCLUSIONS AND IMPLICATIONS Δ9-THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or Δ9-THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB1 receptor-dependent whereas that of Δ9-THC is not. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

Curettage of the epithelium of the vas deferens might be a safe and effective method of male sterilization. We conducted a pilot study of vasectomy by epithelial curettage with a novel microcurette called the Vas-X in 12 normal men requesting elective sterilization. Seminal fluid analysis was obtained monthly after the procedure for 6 months. Pain was assessed by questionnaire. Three months after the procedure, all men attained sperm concentrations of less than 0.2 million sperm per mL, and seven were azoospermic. Post-procedural pain was minimal. Nine men ultimately achieved and maintained azoospermia; however, 4 to 6 months after the procedure, sperm concentrations increased in three of the 12 subjects, necessitating repeat vasectomy. Microscopic examination of the vas deferens from these failures revealed re-canalization. Vasectomy by epithelial curettage can result in effective sterilization; however, 1/4 of the subjects were not effectively sterilized by the procedure due to re-canalization of the vas deferens. Epithelial curettage will require further refinement to determine if it is a viable form of vasectomy.

According to the Americans with Disabilities Act (1990), couples with blood-borne viruses that lead to infectious disease cannot be denied fertility treatment as long as the direct threat to the health and safety of others can be reduced or eliminated by a modification of policies or procedures. Three types of infectious patients are commonly discussed in the context of fertility treatment: those with human immunodeficiency virus (HIV), hepatitis C or hepatitis B. Seventy-five per cent of hepatitis C or HIV positive men and women are in their reproductive years, and these couples look to assisted reproductive techniques for risk reduction in conceiving a pregnancy. In many cases, only one partner is infected. Legal and ethical questions about treatment of infectious patients aside, the question most asked by clinical embryologists and andrologists is: "What are the laboratory protocols for working with gametes and embryos from patients with infectious disease?" The serostatus of each patient is the key that informs appropriate treatments. This guidance document describes protocols for handling gametes from seroconcordant and serodiscordant couples with infectious disease. With minor modifications, infectious patients with stable disease status and undetectable or low viral load can be accommodated in the IVF laboratory.

Our objective was to identify predictors of improved postthaw semen quality in men with testicular cancer banking sperm for fertility preservation. We reviewed 173 individual semen samples provided by 67 men with testicular germ cell tumor (TGCT) who cryopreserved sperm before gonadotoxic treatment between 1994 and 2010 at our tertiary university medical center. Our main outcomes measures were independent predictors for the greater postthaw total motile count (TMC) in men with TGCT. Men with NSGCT were more likely to be younger (P < 0.01) and had high cancer stage (II or III, P < 0.01) compared with men with seminoma. In our multiple regression model, NSGCT histology, use of density gradient purification, and fresh TMC > median fresh TMC each had increased odds of a postthaw TMC greater than median postthaw TMC. Interestingly, age, advanced cancer stage (II or III), rapid freezing protocol, and motility enhancer did not show increased odds of improved postthaw TMC in our models. In conclusion, men with TGCT or poor fresh TMC should consider preserving additional vials (at least 15 vials) before oncologic treatment. Density gradient purification should be routinely used to optimize postthaw TMC in men with TGCT. Larger, randomized studies evaluating cancer stage and various cryopreservation techniques are needed to assist in counseling men with TGCT regarding fertility preservation and optimizing cryosurvival.

Antigen-specific vaccines are one of several molecularly targeted approaches under investigation as possible treatments for prostate cancer. Important to the development of vaccines is the identification of appropriate target antigens. We hypothesized that antigens of the prostate might be identified in patients with the chronic prostatitis/pelvic pain syndrome, a syndrome for which an autoimmune pathology has been proposed. Such antigens might represent naturally recognized target antigens of the prostate that could be investigated in the future as prostate tumor antigens. In this report, we used SEREX to identify proteins expressed in a prostate cDNA expression library recognized by IgG from the sera of patients with chronic prostatitis. Candidate proteins were evaluated using a panel of sera from 62 subjects with symptomatic prostatitis and 71 control male blood donors. We identified one protein that was recognized primarily in sera from subjects with prostatitis compared with controls. MAD-PRO-34, a nucleolar autoantigen, was recognized in 6/62 subjects and 0/71 controls (p = 0.00897). This protein had previously been identified as an autoantigen in patients with prostate cancer. In addition, the NY-CO-7 protein was recognized in 9/62 subjects and 3/71 controls (p = 0.0654). Two subjects had IgG specific for both the MAD-PRO-34 and NY-CO-7 gene products. Our results demonstrate that some patients with the chronic prostatitis/pelvic pain syndrome have autoantibodies to specific proteins. Proteins identified, and MAD-PRO-34 in particular, could be further investigated as potential prostate tumor antigens.

Purpose: Previous reports have identified bacteria in the prostate of men with chronic pelvic pain syndrome. To examine whether prostatic bacteria are more prevalent among patients with chronic pelvic pain syndrome than among those without pelvic pain, we compared 4-glass urine test and prostate biopsy results.Materials and Methods: A total of 120 patients with types IIIa and IIIb chronic pelvic pain syndrome and 60 asymptomatic controls underwent a standard 4-glass urine test, examination of expressed prostatic secretion leukocytes by hemocytometer and transperineal, digitally guided prostate biopsies. Tissue was cultured for aerobes, anaerobes, Trichomonas vaginalis, Chlamydia trachomatis and herpes simplex virus. Skin cultures were performed on a subset of patients and controls.Results: Positive prostate biopsy cultures were obtained from patients and controls. Bacteria were found in 45 of 118 pain patients (38%) and in 21 of 59 controls (36%) (p = 0.74). Older men were more likely to have posi...

Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types.

Purpose We evaluated microscopic methods of detecting inflammation in expressed prostatic secretions. Materials and Methods Methods of counting expressed prostatic secretion leukocytes were compared in 251 samples from 159 patients with chronic prostatitis/chronic pelvic pain syndrome, including traditional wet mounts, hemocytometer derived concentrations and expressed prostatic secretion smears stained with Gram's method or DiffQuick stain (Dade International, Inc., Miami, Florida). Results Of 159 initial patient evaluations 84 (53%) showed inflammation by hemocytometer concentration at 500 leukocytes per mm. 3 or greater but only 37 (23%) were considered inflammation by the traditional wet mount method (p 3 or greater the hemocytometer still identified significantly more patients (41%) and specimens (48%) with inflammation than the wet mount. The hemocytometer method had a substantially lower interassay and intra-assay coefficient of variation than the wet mount method. Polymorphonuclear neutrophils and macrophages were the most common cells observed on stained smears, which detected inflammation in 147 specimens (59%) by DiffQuick but in only 98 (39%) by Gram's method. Conclusions Detecting inflammation in expressed prostatic secretions is method dependent. Significantly more cases of inflammation were detected by hemocytometer than by the traditional wet mount technique. Because the wet mount method also proved more variable than the hemocytometer and highly sensitive to volume, its use is not recommended. These findings support the adoption of hemocytometer and staining methods for accurate evaluation of expressed prostatic secretion inflammation in men with chronic prostatitis/chronic pelvic pain syndrome.

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.

Protein C inhibitor is a heparin-dependent serine protease inhibitor present in plasma at about 0.08 mumol l-1. Protein C inhibitor inhibits activated protein C and other coagulation factors. Previously, we described the presence of high protein C inhibitor levels in human semen (3.1 mumol l-1) and showed potential roles of the inhibitor in human reproduction. Here, we show that protein C inhibitor is present in an active form in follicular fluid at about 0.1 mumol l-1 and that purified, functionally active human plasma-derived and inactive, semen-derived protein C inhibitor and a synthetic peptide derived from its sequence inhibited both binding and penetration of zona-free hamster oocytes by human sperm. The binding inhibition by protein C inhibitor was dose dependent, with 50% inhibition at 0.037 mumol l-1 inhibitor (45 +/- 17 sperm per egg versus 90 +/- 23 in control experiments). The inhibitor also blocked in a dose-dependent manner the penetration of zona-free hamster eggs by human sperm (20 +/- 7% fertilized eggs at 0.1 mumol l-1 protein C inhibitor versus 55 +/- 10% in control experiments). Polyclonal antiprotein C inhibitor or antipeptide antibodies partially abolished the effect of protein C inhibitor and peptide on the inhibition of the binding and penetration of zona-free hamster oocytes by human sperm. The effect of the protein C inhibitor was not dependent on its antiprotease activity since purified semen-derived protein C inhibitor which did not have antiprotease activity gave comparable results. We conclude that protein C inhibitor may be involved in human reproduction at several steps, including the fertilization process.

Purpose: Reactive oxygen species, which are primarily produced by leukocytes, are generally detrimental to sperm. High reactive oxygen species levels are found in men with abnormal sperm function. Since men often have poor sperm characteristics and infertility after vasectomy reversal, fertile men to determine if reactive oxygen species were elevated in the former group.Materials and Methods: We studied semen samples of men with proved fertility (39) and those with previously proved fertility who had undergone vasectomy reversal (45). The presence of leukocytes was determined by Bryan-Leishman staining. Reactive oxygen species endogenous activity was monitored by luminol dependent chemiluminescence in washed cells, including all cells in the semen, and Percoll density gradient purified sperm.Results: After vasovasostomy men had significantly lower sperm concentration, motility and computerized motility measurements than fertile men. Mean reactive oxygen species in washed seminal cells after vasova...

While blood lead concentration has been inversely associated with indicators of reproductive health in occupationally exposed male workers, the utility of lead concentration in semen as an indicator of lead exposure to the male reproductive system has not been fully explored. Blood and semen lead concentrations from 81 lead smelter workers were examined in relation to semen quality and endocrine function parameters. Mean blood and semen lead concentrations were 22.8 μg/dl (range 5–58) and 1.9 μg/dl (range 0.1–17.6), respectively. Total sperm count and concentration decreased with increasing blood lead concentration; p for trend was 0.003 and 0.009, respectively. Semen lead concentration was inversely related to total sperm count (p = 0.05), ejaculate volume (p = 0.001), and serum testosterone (p = 0.004), but not to sperm concentration. The association between semen lead concentration and total sperm count was eliminated when volume was included in the model. Blood lead concentration was more consistently associated with indicators of sperm production than was semen lead. In contrast, semen lead concentration was negatively associated with circulating testosterone concentrations. Our findings indicate that measurement of semen lead may not be a valuable adjunct to conventional blood lead monitoring for investigations of male reproductive system toxicity. Am. J. Ind. Med. 34:464–469, 1998. © 1998 Wiley-Liss, Inc.

Objective To determine whether decreased testicular levels of enzymes necessary for retinoic acid biosynthesis were associated with male infertility, as retinoic acid is known to be necessary for spermatogenesis. Design Observational analysis of testicular tissue samples, sperm indices, and serum hormone concentrations. Setting Two infertility centers in Chile. Patient(s) 32 infertile men and 11 control men. Intervention(s) Measurement of the three enzymes necessary for retinoic acid biosynthesis, aldehyde dehydrogenase (ALDH) 1A1, 1A2, and 1A3, in testicular tissue by a novel liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) peptide assay. Main Outcome Measure(s) ALDH isozyme levels compared by type of infertility and correlated with testicular germ cell numbers, sperm parameters, and serum and intratesticular hormone concentrations. Result(s) Men with infertility had statistically significantly reduced levels of ALDH1A2 but not ALDH1A1 or ALDH1A3 in their testicular tissue compared with men with normal spermatogenesis. The ALDH1A2 protein levels were strongly correlated with the number of germ cells found via testicular biopsy. Conclusion(s) These findings suggest that ALDH1A2 is the enzyme involved in retinoic acid biosynthesis in human germ cells. Further study of the relationship between intratesticular ALDH1A2 and male infertility is warranted to determine whether men with infertility have a reduced ability to synthesize retinoic acid within their germ cells that could impair spermatogenesis.

Advances in diagnostic techniques and therapies have drastically improved cancer survival rates. However, the testes are highly susceptible to chemotherapy and radiation therapy, and spermatogenesis and semen parameters can be negatively impacted either permanently or transiently. Fertility preservation is an important issue to patients, and sperm banking has a positive psychological effect that helps patients cope emotionally with cancer. Innovations in sperm freezing and assisted reproductive technologies are improving the fecundity of males regardless of sperm parameters, and now even allow for fertility potential after gonadotoxic therapy. Experimental options also exist for prepubertal males, and hold promise for use in the future. At present, sperm cryopreservation is underutilized in the setting of a cancer diagnosis, and pretreatment referrals to fertility specialists are inconsistently offered. A multidisciplinary team approach is critical to educate families on the available options and help accomplish fertility preservation in a timely manner.

BACKGROUND Male reproductive potential continues to be adversely affected by many environmental, industrial and pharmaceutical toxins. Pre-emptive testing for reproductive toxicological (side-)effects remains limited, or even non-existent. Many products that come into direct contact with spermatozoa lack adequate testing for the absence of adverse effects, and numerous products that are intended for exposure to spermatozoa have only a general assumption of safety based on the absence of evidence of actual harm. Such assumptions can have unfortunate adverse impacts on at-risk individuals (e.g. couples who are trying to conceive), illustrating a clear need for appropriate up-front testing to establish actual 'sperm safety'. METHODS After compiling a list of general areas within the review's scope, relevant literature and other information was obtained from the authors' personal professional libraries and archives, and supplemented as necessary using PubMed and Google searches. Review by co-authors identified and eliminated errors of omission or bias. RESULTS This review provides an overview of the broad range of substances, materials and products that can affect male fertility, especially through sperm fertilizing ability, along with a discussion of practical methods and bioassays for their evaluation. It is concluded that products can only be claimed to be 'sperm-safe' after performing objective, properly designed experimental studies; extrapolation from supposed predicate products or other assumptions cannot be trusted. CONCLUSIONS We call for adopting the precautionary principle, especially when exposure to a product might affect not only a couple's fertility potential but also the health of resulting offspring and perhaps future generations.

The MYC transcription factor, together with its obligate dimerization partner, MAX, regulates widespread gene expression and is implicated in the etiology of a broad spectrum of cancers. MYC and MAX are members of the MAX-MLX superfamily of bHLHZ transcription factors (which includes MYC, MAX, MXD, MLX, and MONDO/ChREBP). Members of this highly conserved family respond to environmental cues to modulate gene expression programs and control cellular fate. We recently showed that metabolic reprogramming and survival in MYC-driven cancer cells is coordinately regulated by MYC-MAX and MONDOA-MLX. While knockdown of MONDOA or MLX affected multiple metabolic pathways in MYC-induced tumors, we found that decreased fatty acid synthesis primarily accounted for the loss of viability (Carroll et al. Cancer Cell 27:271, 2015) To explore the physiological role of the MLX branch of the MAX-MLX network we generated mice with targeted deletion of Mlx. Mlx null mice appeared to develop normally; however, we found that Mlx null males are infertile, with strikingly diminished sperm production, viability, and motility. MondoA knockout mice exhibited a similar phenotype. By employing tissue-specific Cre-mediated deletion of Mlx, we uncovered metabolic defects in both Sertoli cells as well as germ cells, including loss of lipogenic enzymes and altered lipid content. Moreover, we found that human male germ cell tumors (MGCTs) overexpress MAX-MLX network proteins and undergo apoptosis upon loss of MONDOA/MLX. Importantly, the same metabolic pathways shown to be crucial for survival of neuroblastoma are also required for survival of MGCTs. Furthermore. we have uncovered a critical role for MLX in myokine production required for myoblast fusion in vitro. Moreover, mice bearing homozygous deletion of Mlx fail to regenerate muscle following cardiotoxin treatment (Hunt et al. Genes Dev. 29:2475, 2015). Taken together, our studies suggest a critical role for the MLX arm of the MAX-MLX network in adaptive metabolic responses in tumorigenesis and development. Funding: NIH grants CA57138 (to RNE) and GM055668 (to DEA); ALSAC of St. Jude Children's Research Hospital, the Ellison Medical Foundation, the Glenn Foundation for Medical Research, and the American Federation for Aging Research (AFAR) (to FD). Citation Format: Patrick A. Carroll, Pei-Feng Cheng, Charles H. Muller, Liam Hunt, Fabio Demontis, Don E. Ayer, Robert N. Eisenman. Transcriptional regulation by the MAX-like protein MLX is required for adaptive metabolic responses in development and tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr PL02-02.

An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a steady rate. Addition of antioxidants temporarily interrupts oxygen radical generation, resulting in an inhibition curve. A linear relationship exists between the area of the inhibition curve and the molar quantity of added antioxidant used to quantify total nonenzymatic antioxidant capacity (TAC) in biological fluids including seminal plasma. We streamline the existing enhanced chemiluminescence technique by using a microtiter plate luminometer. A plate luminometer is as accurate as a tube luminometer in measuring TAC, using identical reaction volumes. As little as 1-50 μL of sample may be analyzed. A plate luminometer can detect molar Trolox equivalents as low as 12.5 μM, compared to 25 μM in tube luminometer, using identical volumes. The plate luminometer assay is made even more rapid with use of an injector.

In some cases, human spermatozoa to be used for in vitro fertilization are processed from testicular or epididymal biopsies collected in the clinic or operating room. An appropriately equipped Andrology or Embryology Laboratory is required. Sterility must be maintained at all stages from collection and transport to identification and processing to insemination or cryopreservation. The technologist must be able to properly process and identify spermatozoa from aspirates, seminiferous tubules or pieces of testicular tissue. Recovery of undamaged spermatozoa from tubules or tissue requires mincing, squeezing, or vortexing the tissue, usually without the need of enzymatic digestion. A motility stimulant such as Pentoxifylline is commonly used to calculate the number of functionally competent spermatozoa. After recovery, spermatozoa may be used immediately for IVF-ICSI, incubated overnight prior to IVF-ICSI, or cryopreserved for future use. Methods for identifying, purifying, and determining the number and motility of spermatozoa during these processes are presented.

In some cases, human spermatozoa to be used for in vitro fertilization are processed from testicular or epididymal biopsies collected in the clinic or operating room. An appropriately equipped Andrology or Embryology Laboratory is required. Sterility must be maintained at all stages from collection and transport to identification and processing to insemination or cryopreservation. The technologist must be able to properly process and identify spermatozoa from aspirates, seminiferous tubules or pieces of testicular tissue. Recovery of undamaged spermatozoa from tubules or tissue requires mincing, squeezing, or vortexing the tissue, usually without the need of enzymatic digestion. A motility stimulant such as Pentoxifylline is commonly used to calculate the number of functionally competent spermatozoa. After recovery, spermatozoa may be used immediately for IVF-ICSI, incubated overnight prior to IVF-ICSI, or cryopreserved for future use. Methods for identifying, purifying, and determining the number and motility of spermatozoa during these processes are presented.

An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a steady rate. Addition of antioxidants temporarily interrupts oxygen radical generation, resulting in an inhibition curve. A linear relationship exists between the area of the inhibition curve and the molar quantity of added antioxidant used to quantify total nonenzymatic antioxidant capacity (TAC) in biological fluids including seminal plasma. We streamline the existing enhanced chemiluminescence technique by using a microtiter plate luminometer. A plate luminometer is as accurate as a tube luminometer in measuring TAC, using identical reaction volumes. As little as 1-50 μL of sample may be analyzed. A plate luminometer can detect molar Trolox equivalents as low as 12.5 μM, compared to 25 μM in tube luminometer, using identical volumes. The plate luminometer assay is made even more rapid with use of an injector.

Objective To characterize sperm parameters from thawed semen samples of men with different cancers who cryopreserved semen before oncologic therapy. Design Retrospective cohort study. Setting Tertiary academic medical center. Patient(s) 1,010 semen samples collected between 1994 to 2010. Intervention(s) None. Main Outcome Measure(s) Mean total motile count (TMC), change in percentage motility and percentage survival (100 * [postthaw % motility/raw % motility]) for each cancer compared with data from samples of men without cancer (the "procreative management" group), and proportion of postthaw samples with TMC >5 × 10 6 . Result(s) The procreative management group had the best raw and postthaw semen quality. The best raw and postthaw semen quality for cancer patients occurred in those with prostate cancer (TMC of 155.1 and 53.2 × 10 6 , respectively) and the worst in those with leukemias. Lymphoid leukemias demonstrated the worst raw TMC (26.8 × 10 6 ), but myeloid leukemias displayed the worst postthaw TMC (6.9 × 10 6 ). The testicular cancer group was the only group with a statistically significantly lower chance of having TMC >5 × 10 6 . Conclusion(s) Men with testicular cancer were most commonly referred for sperm cryopreservation and were the only group that was statistically significantly less likely to have TMC >5 × 10 6 on postthaw semen analysis. The most severe reduction in TMC was seen in the myeloid leukemia group, suggesting that these patients along with men with testis cancer and those with lymphoid leukemia should be counseled to provide increased numbers of specimens for fertility preservation.

Objective To identify men with leukocytospermia and prostatitis in an infertility practice and evaluate the effect of various treatments and recurrence rates after treatment. Design A prospective randomized trial of men with leukocytospermia. Setting Academic tertiary infertility clinic. Patients One hundred two men with leukocytospermia identified on smear of semen using Bryan-Leishman stain and in expressed prostatic secretion. Interventions Treatment groups were no treatment group; antibiotic treatment alone group; frequent ejaculation alone group; and antibiotic treatment with frequent ejaculation group. Main Outcome Measure Resolution of leukocytospermia on semen smear. Results Significant resolution of leukocytospermia occurred in all treatment groups at 1 month compared with no treatment. The resolution was sustained at 2 and 3 months only in those who took antibiotics and frequently ejaculated. Conclusions Antibiotic treatment, frequent ejaculation, and antibiotic treatment with frequent ejaculation effectively treat leukocytospermia immediately after the treatment phase. However, only antibiotic treatment coupled with frequent ejaculation is effective 3 months after treatment.

Fragmentary evidence indicates that intracellular [Ca2+] (Cai) mediates sperm chemotaxis. However, neither correlations of swimming responses to chemoattractant-induced alterations of Cai nor explanations of how chemoattractant gradients control Cai exist. Here Cai increases produced by the egg peptide speract-not previously known to cause flagellar responses--were prolonged by treatment with 3-isobutyl-1-methylxanthine (IBMX). Flagellar waveform asymmetry then increased 40% and swimming paths became tightly circular. Moreover, both responses required external Ca2+ (as does sperm chemotaxis to eggs and egg products). Cai increases by the established chemotactic peptide resact also required external Ca2+ and were enhanced by IBMX. Therefore, diverse egg peptides may use fundamentally similar mechanisms to control Cai and thereby swimming behavior in chemotaxis. Repetitive increasing additions of speract produced adaptive membrane potential and Cai responses indicating that sperm can detect increasing gradients of egg peptide over a broad concentration range. We offer a model in which shallow or decreasing gradients elevate Cai and redirect swimming paths but sufficiently steep gradients keep Cai low and swimming linear until the egg is reached. A negative-feedback loop, initiated by cGMP-mediated activation of sperm K+ channels and terminated by subsequent inactivation of guanylyl cyclase, may coordinate gradient detection with control of Cai. Continued stimulation of more receptors by steeply increasing gradients of egg peptide thus maintains membrane hyperpolarization and suppresses Ca2+ entry and Cai elevation. The molecular basis for chemotaxis therefore is explained as translation of the spatial gradient of peptide concentration into changes in K+ channel activity in the time domain.

Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB(1) cannabinoid receptor agonist, and Δ(9) -tetrahydracannabinol (Δ(9) -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of Δ(9) -THC on male fecundity were determined.Effects of Δ(9) -THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of Δ(9) -THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by Δ(9) -THC-treated males. KEY RESULTS Pretreatment of sperm for 15 min with 1 µM Δ(9) -THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 µM WIN 55,212-2 or 10 µM Δ(9) -THC for 30 min reduced sperm ATP levels. In sperm lacking CB(1) receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of Δ(9) -THC persisted. Administration of 50 mg·kg(-1) Δ(9) -THC to male mice just before mating caused a 20% decrease in embryonic litter size.Δ(9) -THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or Δ(9) -THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB(1) receptor-dependent whereas that of Δ(9) -THC is not. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Objective To determine if one mechanism of albumin-mediated support of human sperm capacitation is lipid (cholesterol) transfer activity and contamination of albumin with Lipid Transfer Protein-I (LTP-I). Design and Main Outcome Measures Measure lipid transfer activity in various bovine and human albumin preparations; relate this activity to albumin-supported capacitation (measured by zona-free hamster oocyte sperm penetration assay) and acrosome reactions; and attempt to detect LTP-I in active albumins. Remove LTP-I from albumin which supports capacitation and reassess this support. Reconstitute capacitation support by addition of purified LTP-I. Setting and Subjects Healthy sperm donors with normal semen analyses were recruited by the Reproductive Biology-Andrology Laboratory in a university medical center. Results Albumin preparations that effectively support capacitation have high levels of lipid transfer activity and of LTP-I, a protein responsible for lipid transfer activity. Preparations with lower levels of capacitation support have less lipid transfer activity. Removal of LTP-I from supportive albumin significantly reduces the capacitation support, and this is restored by purified LTP-I. Progesterone concentrations in these preparations are negligible. Conclusions The variable abilities of albumin preparations to support in vitro sperm capacitation are largely dependent on the presence of contaminating LTP-I. The cholesterol transfer activity of this protein, which is present in human serum and follicular fluid, may be one mechanism in the process of capacitation.

The bisdichloroacetyldiamine WIN 18,446 reversibly inhibits spermatogenesis in many species, including humans; however, the mechanism by which WIN 18,446 functions is unknown. As retinoic acid is essential for spermatogenesis, we hypothesized that WIN 18,446 might inhibit retinoic acid biosynthesis from retinol (vitamin A) within the testes by inhibiting the enzyme aldehyde dehydrogenase 1a2 (ALDH1a2). We studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro, and on spermatogenesis and fertility in vivo, in mature male rabbits for 16 weeks. WIN 18,446 markedly inhibited ALDH1a2 enzyme activity in vitro with an IC(50) of 0.3 μM. In vivo, the oral administration of 200 mg/kg WIN 18,446 to male rabbits for 16 weeks significantly reduced intratesticular concentrations of retinoic acid, severely impaired spermatogenesis, and caused infertility. Reduced concentrations of intratesticular retinoic acid were apparent after only 4 weeks of treatment and preceded the decrease in sperm counts and the loss of mature germ cells in tissue samples. Sperm counts and fertility recovered after treatment was discontinued. These findings demonstrate that bisdichloroacetyldiamines such as WIN 18,446 reversibly suppress spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2. These findings suggest that ALDH1a2 is a promising target for the development of a reversible, nonhormonal male contraceptive.