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Purpose: The use of antioxidants is common practice in the management of infertile patients. However, there are no estab-lished guidelines by professional societies on antioxidant use for male infertility. Materials and Materials and Methods:Methods: Using an online survey, this study aimed to evaluate the practice pattern of reproductive specialists to determine the clinical utility of oxidative stress (OS) testing and antioxidant prescriptions to treat male infertility. Results:Results: Responses from 1,327 participants representing 6 continents, showed the largest participant representation being from Asia (46.8%). The majority of participants were attending physicians (59.6%), with 61.3% having more than 10 years of experience in the field of male infertility. Approximately two-thirds of clinicians (65.7%) participated in this survey did not order any diagnostic tests for OS. Sperm DNA fragmentation was the most common infertility test beyond a semen analysis that was prescribed to study oxidative stress-related dysfunctions (53.4%). OS was mainly tested in the presence of lifestyle risk factors (24.6%) or sperm abnormalities (16.3%). Interestingly, antioxidants were prescribed by 85.6% of clinicians, for a duration of 3 (43.7%) or 3?? months (38.6%). A large variety of antioxidants and dietary supplements were prescribed, and scientific evidence were mostly considered to be modest to support their clinical use. Results were not influenced by the physician?셲 age, geographic origin, experience or training in male infertility. Conclusions:Conclusions: This study is the largest online survey performed to date on this topic and demonstrates 1) a worldwide under-standing of the importance of this therapeutic option, and 2) a widely prevalent use of antioxidants to treat male infertility. Finally, the necessity of evidence-based clinical practice guidelines from professional societies is highlighted.

Oxidative stress is prevalent among infertile men and is a significant cause of sperm DNA damage. Since sperm DNA damage may reduce embryo quality and increase miscarriage rates, it is possible that untreated sperm oxidative stress may impair in vitro fertilization (IVF) live birth rates. Given that the antioxidant Menevit is reported to reduce sperm DNA damage, it was hypothesized that men's consumption of this supplement may alter IVF outcomes. Therefore, a retrospective cohort study was conducted analyzing outcomes for couples undergoing their first fresh embryo transfer. Men were classified as controls if they were taking no supplements, health conscious controls if taking “general health” supplements, or Menevit users. Men with karyotype abnormalities, or cycles using donated, frozen and surgically extracted sperm were excluded. Among the final study cohort of 657 men, live birth rates were significantly higher in Menevit users than controls (multivariate adjusted odds ratio [OR]: 1.57, 95% confidence interval [CI]: 1.01–2.45, P= 0.046), but not between controls taking no supplements and those using general health supplements, thereby suggesting that potential health conscious behavior in supplement users is unlikely responsible for the superior outcomes in Menevit users. Interestingly, in a post hoc sensitivity analysis, live birth rates among Menevit users were statistically superior to controls for lean men (OR: 2.73, 95% CI: 1.18–6.28; P= 0.019), not their overweight/obese counterparts (OR: 1.29, 95% CI: 0.75–2.22, P = 0.37). The results of this large cohort study therefore support a positive association between men's use of the Menevit antioxidant during IVF treatment and live birth rates, especially in lean individuals. Refereed/Peer-reviewed

Does increased body mass index (BMI) without an underlying metabolic issue negatively influence semen quality? Proof of concept we conducted retrospective data analysis of men (N = 84) undergoing assisted reproductive technology, who had liver function testing with fasted glucose concentrations and corresponding hormone profile (testosterone, LH, FSH and prolactin) and semen analysis. Sperm count and total concentration were only reduced in metabolically unhealthy overweight/obese men. Serum GTT was the biggest predictor of Normozoospermia and Oligospermia, with BMI having no effect. Increased BMI without an underlying metabolic condition (in particular signs of NAFLD) has no influence on semen quality.

Study question Can in couples with unexplained infertility a prognosis-tailored management strategy, that delays treatment if natural conception prospects are good, reduce costs without affecting live-birth rate? Summary answer In couples with unexplained infertility, use of a prognostic tool for natural conception followed by expectant management in good-prognosis couples is cost-effective. What is known already Few countries have guidelines for the assessment of the likelihood of natural conception to determine access to publicly funded ART. In the Netherlands and New-Zealand, couples with unexplained infertility who have a good prognosis for natural conception are encouraged to delay starting ART. However, the cost-effectiveness of this prognosis-tailored treatment strategy has not been determined. Study design, size, duration We studied couples with unexplained infertility to compare a prognosis-tailored strategy to care-as-usual. In the prognosis-tailored strategy, couples were assessed using Hunault’s prediction model. In good-prognosis couples (12-months natural conception >40%), outcomes without ART were modelled by censoring observations after start of ART. We then assumed that poor-prognosis couples ( Participants/materials, setting, methods We studied 272 couples with unexplained infertility. Costs of in vitro fertilisation (IVF) and intra-uterine insemination (IUI) were calculated based on the out-of-pocket costs and Australian Medicare costs. In a cost-effectiveness model, we compared costs and effects of both strategies. Main results and the role of chance The prognostic model classified 272 couples with unexplained infertility as favourable (N = 107 (39.3%) or unfavourable prognosis (N = 165 (60.7%)) for natural conception. In the prognosis-tailored strategy, the cumulative live-birth rate was 71.1% (95% CI 64.7% - 76.4%) while the number of ART cycles was 393 (353 IVF; 40 IUI). In care-as-usual strategy, the cumulative conception rate leading to live-birth for the cohort of 272 couples, who underwent a total of 398 IVF cycles and 48 IUI cycles, was 72.1% (95% CI 65.7% - 77.4%). Mean time to conception leading to live birth was 388 days in the prognosis-tailored strategy and 419 days in the care-as-usual strategy. This translated for the 272 couples into potential savings of 45 IVF cycles and eight IUI cycles, which cost a total of AUD$ 125,817 for out-of-pocket and AUD$ 264,497 for Australian Medicare. The average cost savings per couple was AUD$ 1,435 (out-of-pocket AUD$ 463 per couple and Australian Medicare AUD$ 962 per couple). The incremental cost-effectiveness ratio, which was calculated as the total costs per additional live-births, was AUD$ 143,497 per additional live birth. Limitations, reasons for caution This study was limited to couples at a single IVF clinic. The modelling was also based on several key assumptions, particularly the number of fresh and frozen embryo transfer cycles for each couple. Wider implications of the findings: Our results show that in couples with unexplained infertility the use of a prognostic model guiding the start of an IVF-treatment reduces costs without compromising live birth rates. Trial registration number Not applicable

Research question What is the predictive value of serum anti-Mullerian hormone (AMH) level for natural conception and its clinical impact on subfertile couples? Design This is a retrospective cohort of ovulatory women seeking fertility consultation in a private fertility clinic. Couples who had an immediate indication for in vitro fertilisation (IVF) were excluded. All natural conceptions leading to live birth before the start of assisted reproduction technology (ART) were followed within 12 months since initial consultation. A prediction model was developed by updating the Hunault model with serum AMH to predict the probabilities of achieving a natural conception leading to live birth. Results There were 325 couples included in the final analysis. The cumulative natural conception rate leading to live birth within 12 months was 20.9% (95% Confidence Interval (CI): 12.9% - 28.2%). The categorical net reclassification improvement (NRI) of AMH is 7.6%. For couples with a predicted chance of natural conception changed from poor ( 30%) by the reference model to good ( ≥ 30%) by the updated model, the cumulative natural conception rate leading to live birth was 52.0%. For couples who had predicted chance of natural conception changed from good to poor by the updated model, the rate was 18.9%. Conclusions Adding serum AMH to the routine fertility work-up may improve prognosis-based treatment policy and help to prevent unnecessary costs and stress for couples. Prospective validation of the updated model with AMH is required before clinical application.

International audience; Retrograde ejaculation (RE) is a condition defined as the backward flow of the semen during ejaculation, and when present can result in male infertility. RE may be partial or complete, resulting in either low seminal volume or complete absence of the ejaculate (dry ejaculate). RE can result from anatomic, neurological or pharmacological conditions. The treatment approaches outlined are determined by the cause. Alkalinizing urinary pH with oral medications or by adding sperm wash media into the bladder prior to ejaculation may preserve the viability of the sperm. This article provides a step-by-step guide to diagnose RE and the optimal techniques to retrieve sperm. Copyright

Background Insulin-like peptide 3 (INSL3) is a constitutive, secreted peptide produced in the male uniquely by the Leydig cells of the testes. It is a biomarker for Leydig cell functional capacity, which is a measure of the numbers and differentiation status of these steroidogenic cells and lacks the biological and technical variance of the steroid testosterone. This retrospective study was carried out to examine the relationship between seminal parameters and the Leydig cell compartment, and secondarily to assess other factors responsible for determining Leydig cell functional capacity. Methods INSL3 was assessed together with seminal, anthropometric, and hormonal parameters in a Swedish cohort of 18-year-old men, representing the average population, and in a smaller, more heterogeneous cohort of men visiting an Australian infertility clinic. Results and discussion Average INSL3 concentration at 18 years is greater than that reported at younger or older ages and indicated a large 10-fold variation. In neither cohort was there a relationship between INSL3 concentration and any semen parameter. For the larger, more uniform Swedish cohort of young men, there was a significant negative relationship between INSL3 and BMI, supporting the idea that adult Leydig cell functional capacity may be established during puberty. In both cohorts, there was a significant relationship between INSL3 and FSH, but not LH concentration. No relationship was found between INSL3 and androgen receptor trinucleotide repeat polymorphisms, reinforcing the notion that Leydig cell functional capacity is unlikely to be determined by androgen influence alone. Nor did INSL3 correlate with the T/LH ratio, an alternative measure of Leydig cell functional capacity, supporting the view that these are independent measures of Leydig cell function.

Research question: Is PIEZO-intracytoplasmic sperm injection (ICSI) coupled with a new novel operational fluid (perfluoro-n-octane) superior to standard ICSI? Design: A cohort of patients (n = 69) undertaking microinjection were recruited between January and November 2019 and were then prospectively case-matched. Patients required six or more mature oocytes for inclusion in the study. PIEZO-ICSI uses high-speed microinjection drilling to penetrate the zona and oolemma and deposit the spermatozoa into the cytoplasm, compared with the traditional ‘cutting’ action of ICSI. The primary outcome was fertilization, with secondary outcomes including oocyte degeneration, abnormal fertilization, embryo cryopreservation and embryo utilization. Results: PIEZO-ICSI resulted in significantly higher fertilization rates (80.5 ± 2.4% vs 65.8 ± 2.3%, P < 0.0001) and lower oocyte degeneration rates (4.4 ± 1.3% vs 8.6 ± 1.2%, P = 0.019) and abnormal fertilization rates (2.9 ± 1.1% vs 7.4 ± 1.1%; P = 0.003) compared with standard ICSI. This improvement in fertilization was of most benefit in patients aged ≥38 years. This increase in fertilization increased the number of good quality embryos that were available for cryopreservation/transfer (3.8 ± 0.2 vs 3.1 ± 0.2; P = 0.038), such that patients on average had one extra usable embryo per cycle compared with standard ICSI. There were no differences to Day 5 embryo development or clinical pregnancy from fresh embryo transfer (57.1% PIEZO-ICSI vs 60.0% ICSI) between microinjection methods, although pregnancy outcomes were underpowered. Conclusions: PIEZO-ICSI significantly increased fertilization rates, thereby increasing the number of embryos available for cryopreservation compared with standard ICSI. Further prospective studies assessing cumulative pregnancy rates are warranted Refereed/Peer-reviewed

PURPOSE: To determine if the use of ICSI in women of advanced maternal age with non-male factor infertility increases chances of live birth. METHODS: Retrospective data analysis of 10 years of cycle data from a single Australian IVF clinic (Repromed). First cycle patients only of an advanced maternal age (≥ 35 years) with non-male factor infertility utilising standard IVF or ICSI insemination and having at least three oocytes collected at egg pick up were assessed for live birth following transfer of single genetically unscreened blastocyst (N = 577). Subanalysis of clinical pregnancy, miscarriage, fertilisation, embryo utilisation rate and having a blastocyst for transfer were considered. Unadjusted, covariate adjusted and propensity score weighted analysis were performed. RESULTS: The use of standard IVF insemination in women ≥ 35 years with non-male factor infertility increased the chance of a live birth compared with ICSI insemination (unadjusted OR = 2.72, 95% CI [1.78, 4.17]; adjusted OR = 2.64, 95% CI [1.64, 4.27] and weighted OR = 2.26, 95% CI [1.72, 2.98] 31% vs 14%). All other outcomes (fertilisation rate, embryo utilisation, blastocyst for embryo transfer and miscarriage rate) were unaffected. CONCLUSION: In couples with advanced maternal age and non-male factor infertility, standard IVF insemination appears to increase the chance of a live birth compared with ICSI. As such, the results of this study support the use of routine IVF as the preferred insemination technique for older women in non-male factor infertility. However, future randomised controlled trials are still required to assess this policy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-020-02026-8.

Objectives Intravenous infusion of Intralipid is an adjunct therapy in assisted reproduction treatment (ART) when immune‐associated infertility is suspected. Here, we evaluated the effect of Intralipid infusion on regulatory T cells (Treg cells), effector T cells and plasma cytokines in peripheral blood of women undertaking IVF. Methods This prospective, observational pilot study assessed Intralipid infusion in 14 women exhibiting recurrent implantation failure, a clinical sign of immune‐associated infertility. Peripheral blood was collected immediately prior to and 7 days after intravenous administration of Intralipid. Plasma cytokines were measured by Luminex, and T‐cell subsets were analysed by flow cytometry. Results A small increase in conventional CD8+ T cells occurred after Intralipid infusion, but no change was seen in CD4+ Treg cells, or naïve, memory or effector memory T cells. Proliferation marker Ki67, transcription factors Tbet and RORγt, and markers of suppressive capacity CTLA4 and HLA‐DR were unchanged. Dimensionality‐reduction analysis using the tSNE algorithm confirmed no phenotype shift within Treg cells or other T cells. Intralipid infusion increased plasma CCL2, CCL3, CXCL8, GM‐CSF, G‐CSF, IL‐6, IL‐21, TNF and VEGF. Conclusion Intralipid infusion elicited elevated pro‐inflammatory cytokines, and a minor increase in CD8+ T cells, but no change in pro‐tolerogenic Treg cells. Notwithstanding the limitation of no placebo control, the results do not support Intralipid as a candidate intervention to attenuate the Treg cell response in women undergoing ART. Future placebo‐controlled studies are needed to confirm the potential efficacy and clinical significance of Intralipid in attenuating cytokine induction and circulating CD8+ T cells., The lipid emulsion Intralipid is commonly used as an adjunct therapy in assisted reproduction, but whether and how it improves immune adaptation for pregnancy is unknown. Here, we showed Intralipid infusion is associated with elevated plasma levels of several embryotrophic cytokines that may promote implantation, but no change in the abundance or phenotypes of regulatory T cells that mediate maternal immune tolerance.

International audience; The current WHO 2010 manual for human semen analysis defines leukocytospermia as the presence of peroxidase-positive leukocytes at a concentration >1×106/mL of semen. Granular leukocytes when activated are capable of generating high levels of reactive oxygen species in semen resulting in oxidative stress. Oxidative stress has been correlated with poor sperm quality, increased level of sperm DNA fragmentation and low fertility potential. The presence of leukocytes and pathogens in the semen may be a sign of infection and/or localized inflammatory response in the male genital tract and the accessory glands. Common uro-pathogens including Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, and Escherichia coli can cause epididymitis, epididymo-orchitis, or prostatitis. The relationship between leukocytospermia and infection is unclear. Therefore, we describe the pathogens responsible for male genital tract infections and their association with leukocytospermia. The review also examines the diagnostic tests available to identify seminal leukocytes. The role of leukocytospermia in male infertility and its management is also discussed.

BACKGROUND The complications associated with in vitro fertilisation (IVF) for both the offspring and mother, and its high cost make it essential to tailor the technology to those infertile couples who truly benefit from it. AIMS To determine whether a simple prognostic algorithm could discriminate between couples who require immediate fertility treatments and couples in whom less invasive strategies should be offered first. MATERIALS AND METHODS In this retrospective cohort study, couples were classified into six groups based on the medical necessity of IVF and their prognosis for natural conception: (i) tubal/severe semen factor mandating immediate IVF due to the very low chance of natural conception; (ii) pure anovulation infertility; (iii) female age ≥39 years; and (iv) unexplained/mild male infertility (no indication for immediate treatment) with (4A) good, (4B) moderate or (4C) poor prognosis of natural conception, as per an existing, validated prognostic model. For each group, we constructed Kaplan-Meier curves to measure natural conception and the effect of fertility treatment. RESULTS The 12 months cumulative live birth rate for couples with unexplained or mild male infertility and poor prognosis increased from 1% without treatment to 35% after treatment (P

Objective High-fat diet (HFD)-induced metabolic disorders can lead to impaired sperm production. We aim to investigate if HFD-induced gut microbiota dysbiosis can functionally influence spermatogenesis and sperm motility. Design Faecal microbes derived from the HFD-fed or normal diet (ND)-fed male mice were transplanted to the mice maintained on ND. The gut microbes, sperm count and motility were analysed. Human faecal/semen/blood samples were collected to assess microbiota, sperm quality and endotoxin. Results Transplantation of the HFD gut microbes into the ND-maintained (HFD-FMT) mice resulted in a significant decrease in spermatogenesis and sperm motility, whereas similar transplantation with the microbes from the ND-fed mice failed to do so. Analysis of the microbiota showed a profound increase in genus Bacteroides and Prevotella, both of which likely contributed to the metabolic endotoxaemia in the HFD-FMT mice. Interestingly, the gut microbes from clinical subjects revealed a strong negative correlation between the abundance of Bacteroides-Prevotella and sperm motility, and a positive correlation between blood endotoxin and Bacteroides abundance. Transplantation with HFD microbes also led to intestinal infiltration of T cells and macrophages as well as a significant increase of pro-inflammatory cytokines in the epididymis, suggesting that epididymal inflammation have likely contributed to the impairment of sperm motility. RNA-sequencing revealed significant reduction in the expression of those genes involved in gamete meiosis and testicular mitochondrial functions in the HFD-FMT mice. Conclusion We revealed an intimate linkage between HFD-induced microbiota dysbiosis and defect in spermatogenesis with elevated endotoxin, dysregulation of testicular gene expression and localised epididymal inflammation as the potential causes. Trial registration number NCT03634644.

Oxidative stress is a well-established cause of male infertility. Reactive oxygen species (ROS), produced by sperm and activated leukocytes within the male reproductive tract, are present at higher concentrations in infertile men’s semen and have been conclusively shown to impair sperm function leading to infertility and possible long-term health consequences for any child conceived. In this chapter, we critically analyse the published studies that have attempted to treat male infertility by the reduction of leukocyte production of ROS (anti-inflammatory action), the direct neutralization of ROS by antioxidants or the fortification of sperm against ROS attack. Overall, there is reasonably good evidence that some antioxidant and anti-inflammatory therapies can significantly reduce oxidative damage to sperm DNA and improve sperm membrane function (motility and sperm fertilization capacity). However, what is still contentious is whether these improvements in sperm quality translate into an increase in natural conception rates for infertile couples. While several relatively small studies have shown some antioxidant therapies to assist both in vivo and in vitro conception, larger more definitive studies will be required before antioxidant therapy becomes an established medical practice for infertile males.

The Limulus amebocyte assay (LAL) is increasingly used to quantify metabolic endotoxemia (ME), particularly in feeding studies. However, the assay was not intended to assess plasma at levels typically seen in ME. We aimed to optimize and validate the LAL assay under a range of pre-treatment conditions against the well-established lipopolysaccharide binding protein assay (LBP). Fifteen healthy overweight and obese males aged 28.8 ± 9.1years provided plasma. The LAL assay employed a range of pre-treatments; 70 ◦C for 15 and 30 min and 80 ◦C for 15 and 30 min, ultrasonication (70 ◦C for 10 min and then 40 ◦C for 10 min), and dilution (1:50, 1:75, 1:100, and 1:200 parts) or diluted using 0.5% pyrosperse. Seventeen different plasma pre-treatment methods employed prior to the use of the LAL analytical technique failed to show any relationships with either LBP, or body mass index (BMI; obesity), the biological trigger for ME (p > 0.05 for all). As expected, BMI positively correlated with LBP (r = 0.523, p = 0.052. No relationships were observed between LAL with any of the sample pre-treatments and LBP or BMI. In its current form, the LAL assay is unsuitable for detecting levels of endotoxin typically seen in ME. Refereed/Peer-reviewed

Seminal fluid interacts with the female reproductive tract to initiate a permissive immune response that facilitates embryo implantation and pregnancy success. The immune-regulatory cytokine interferon-gamma (IFNG), which can be elevated in seminal plasma, is associated with reduced fertility. Here, we investigated how IFNG influences the female immune response to seminal fluid. In human Ect1 cervical epithelial cells, IFNG added at physiologically relevant concentrations substantially impaired seminal plasma-induced synthesis of key cytokines colony-stimulating factor 2 (CSF2) and interleukin-6 (IL6). Seminal fluid-induced CSF2 synthesis was also suppressed in the uterus of mice in vivo, when IFNG was delivered transcervically 12 h after mating. Transforming growth factor B1 (TGFB1) is the major seminal fluid signaling factor which elicits CSF2 induction, and IFNG exhibited potent dose-dependent suppression of CSF2 synthesis induced by TGFB1 in murine uterine epithelial cells in vitro. Similarly, IFNG suppressed TGFB1-mediated CSF2 induction in Ect1 cells and human primary cervical epithelial cells; however, IL6 regulation by IFNG was independent of TGFB1. Quantitative PCR confirmed that CSF2 regulation by IFNG in Ect1 cells occurs at the gene transcription level, secondary to IFNG suppression of TGFBR2 encoding TGFB receptor 2. Conversely, TGFB1 suppressed IFNG receptor 1 and 2 genes IFNGR1 and IFNGR2. These data identify IFNG as a potent inhibitor of the TGFB-mediated seminal fluid interaction with relevant reproductive tract epithelia in mice and human. These findings raise the prospect that IFNG in the male partner's seminal fluid impairs immune adaptation for pregnancy following coitus in women. Refereed/Peer-reviewed

Obesity is associated with a reduction in fertility treatment success and increased risks to mother and child. Therefore guidelines of the Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) suggest that a body mass index exceeding 35 kg/m(2) should be an absolute contraindication to assisted fertility treatment such as in vitro fertilisation IVF. In this paper we challenge the ethical and scientific basis for such a ban. Livebirth rates for severely obese women are reduced by up to 30%, but this result is still far better than that observed for many older women who are allowed access to IVF. This prohibition is particularly unjust when IVF is the only treatment capable of producing a pregnancy, such as bilateral tubal blockage or severe male factor infertility. Furthermore, the absolute magnitude of risks to mother or child is relatively small, and while a woman has a right to be educated about these risks, she alone should be allowed to make a decision on proceeding with treatment. We do not prohibit adults from engaging in dangerous sports, nor do we force parents to vaccinate their children, despite the risks. Similarly, we should not prohibit obese women from becoming parents because of increased risk to themselves or their child. Finally, prohibiting obese women's access to IVF to prevent potential harms such as 'fetal programing' is questionable, especially when compared to that child never being born at all. As such, we believe the RANZCOG ban on severely obese women's access to assisted reproductive treatment is unwarranted and should be revised.

The prevailing legal position and opinion of professional societies such as the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine is that posthumous sperm conception should only occur in the presence of explicit written consent from the deceased man. However, in our opinion this is an impractical approach as the majority of deaths of reproductive-age men are sudden and unexpected, thereby precluding explicit consent. Previously in this journal we have outlined arguments supporting a move to a standard of presumed consent for posthumous conception, with provisions for men to 'opt out' and safeguards to protect the welfare of the prospective mother and her child. In a recent commentary in this journal, Kroon outlines arguments against our position of presumed consent as an unacceptable violation of the deceased's autonomy. However, such arguments on the primacy of the rights of the dead are in our opinion not paramount, especially since this position blocks access to posthumous conception for the majority who support its use. The objective of this commentary is to provide a rebuttal to the concerns raised by Kroon and hopefully reorientate the discussion towards the rights and welfare of the living (widow, prospective child), not the dead.

Obesity is known to be associated with impaired testicular function potentially resulting in androgen deficiency and subfertility. While the underlying cause of obesity-related male hypogonadism is multi-factorial, here, we investigated the impact of dietary fat on testicular endocrine function. Ingestion of a high-fat “fast food” mixed meal, a common practice for obese men, produced a 25% fall in serum testosterone within an hour of eating, with levels remaining suppressed below fasting baseline for up to 4 hr. These changes in serum testosterone were not associated with any significant changes in serum gonadotrophins. The nadir in serum testosterone preceded the post-prandial increase in serum IL-6/IL-17 by several hours, suggesting that inflammation was unlikely the cause. Furthermore, intravenous administration of fat (Intralipid) had no impact on testosterone levels, while an identical oral dose of fat did suppress testosterone. These results suggest that fat does not directly impair Leydig cell function, but rather the passage of fat through the intestinal tract elicits a response that indirectly elicits a post-prandial fall in testosterone. Refereed/Peer-reviewed

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

Background: Obesity is known to be associated with inflammation, oxidative stress and a resulting reduction in sperm DNA integrity. Importantly, obesity is also reported to be associated with an increase in intestinal permeability with the passage of intestinal bacteria into the circulation (metabolic endotoxemia) that triggers a systemic state of inflammation and resultant oxidative stress. Therefore, we hypothesised that this obesity related increase in intestinal permeability and resultant metabolic endotoxemia (ME) may activate inflammation within the male reproductive tract, leading to increased reactive oxygen species production, sperm oxidative stress and a decline in DNA integrity. Results: Our pilot study of 37 infertile men confirmed a significant positive correlation between body mass index (BMI), increased intestinal permeability (serum zonulin), metabolic endotoxaemia (LBP), sperm DNA oxidative damage (seminal 8 OhDG) and increasing levels of sperm DNA fragmentation (Halosperm). Metabolic endotoxemia was positively correlated with increasing levels of sperm DNA oxidative damage with this relationship remaining significant, even after adjustment for relevant confounders such as age, BMI and days of abstinence. These observations suggest that metabolic endotoxemia and its associated oxidative stress may be a key driver of sperm DNA damage in obese men. Conclusion: This study confirms a link between obesity, increasing intestinal permeability and endotoxin exposure, and oxidative mediated sperm DNA damage. This warrants further investigation to fully understand the effect of metabolic endotoxemia on male reproductive function which could result in the new therapies to improve male fertility potential. Refereed/Peer-reviewed

STUDY QUESTION Does the rate of miscarriage increase in the setting of adenomyosis independent of other known risk factors for miscarriage such as maternal age, BMI, embryo genetic status? SUMMARY ANSWER Adenomyosis and high BMI both significantly increase miscarriage risk independent of each other, maternal age and embryo health. This study is the first to suggest that ultra-long down regulation GnRH agonist treatment may reduce the rate of early pregnancy loss in adenomyosis patients. WHAT IS KNOWN ALREADY The presence of adenomyosis is known to be associated with lower rates of successful implantation and increased risk of early pregnancy loss. However, it is presently unclear whether this reproductive impairment is directly mediated by adenomyosis itself, or indirectly caused by adenomyosis association with known risk factors for miscarriage such as obesity and advancing maternal age/foetal aneuploidy. STUDY DESIGN, SIZE, DURATION A retrospective cohort study was undertaken in a private infertility (IVF) clinic examining the outcome for women (n = 345) undergoing the transfer of a genetically screened frozen–thawed embryo between 2012 and 2015. PARTICIPANTS/MATERIALS, SETTING AND METHOD A total of 171 women who successfully conceived (positive serum βhCG) following the transfer of a single euploid good morphology frozen–thawed embryo were included in analysis after meeting the inclusion criteria. Only the first conception cycle for each patient was included in the study. Patients with known pre-existing medical risk factors for miscarriage (e.g. thrombophilia, poorly controlled diabetes, coeliac disease, SLE, uterine septum, chromosomal abnormalities) and those women undergoing treatment using donated oocytes and surrogacy were excluded. Patients were then classified as having adenomyosis or not based on a high-quality pelvic ultrasound or MRI. The direct and indirect effects of adenomyosis and BMI on overall miscarriage rate by 12 weeks gestation was then assessed using multivariate logistic regression and mediation analysis. Furthermore, the data were also analysed to elucidate the influence of GnRH ultra-long down-regulation therapy on miscarriage rates. MAIN RESULTS AND ROLE OF CHANCE Overall, the adjusted rate of miscarriage was higher in those patients with adenomyosis compared to those without (44.1 vs 15.3%, P < 0.0001), with most of these miscarriages occurring at the early biochemical stage. The rate of miscarriage was especially high in adenomyosis patients not receiving GnRH agonist pre-treatment (82.4%), compared to those patients who did receive GnRH pre-treatment (35.7%, P = 0.0089). LIMITATIONS, REASONS FOR CAUTION The study is mainly limited by its small sample size and retrospective design which carries inherent potential for bias (i.e. misclassification and errors due to inadequate clinical notes). The small sample size precluded analysis to distinguish how the extent of adenomyosis disease may modify miscarriage risk (i.e. focal or diffuse disease). Furthermore, the relatively low number of adenomyosis patients not receiving GnRH agonist treatment, plus the non-randomized nature of the decision not to offer such treatment, precludes definitive conclusions on the benefit of GnRH agonist therapy to reduce miscarriage risk. WIDER IMPLICATIONS OF THE FINDINGS Considering the significant emotional and financial impact of miscarriage, we suggest screening of all women undergoing IVF treatment for the presence of adenomyosis, with consideration given to ultra-long down regulation GnRH agonist treatment in any woman identified as having adenomyosis. Furthermore, given the persistent and often progressive nature of the disease, adenomyosis should also be considered as a potential uterine cause of recurrent miscarriage. Finally, we hope our study highlights the need for high-quality prospective RCT to be undertaken to provide superior evidence for the potential benefit of GnRH agonist pre-treatment. STUDY FUNDING/COMPETING INTEREST(S) K.T. is a practicing IVF gynaecologist and holds a minority stake in the publicly listed company Monash IVF. The other authors declare that they have no conflict of interest. This study was financially supported by Flinders University Medical School.

Oxidative damage to sperm, with a reduction in fertility and the potential for adverse health consequences for the resulting child, occurs when the production of reactive oxygen species (ROS) by sperm and seminal leukocytes exceeds the neutralization capacity of protective antioxidants contained within seminal plasma, epididymal secretions, and sperm. In order to optimize sperm health and reproductive outcomes, oxidative stress (OS) should be minimized by a combination of improvements in the patient's lifestyle, modification of disease processes known to aggravate oxidative attack, plus the use of antioxidant supplements. In the in vitro fertilization laboratory, careful handling of sperm to minimize the production of ROS while maximizing antioxidant protection is the key to optimal sperm health. This chapter will discuss in detail how clinicians can recognize and manage the key sources of OS, plus practical ways in which sperm health can be optimized by the clinical scientist (embryologist) in the laboratory.

Previous observational studies have suggested that β-cryptoxanthin, a micronutrient present in yellow and orange fruit and vegetables, may help delay the onset of menopause. Given the widespread social trend of delaying pregnancy, the possibility that β-cryptoxanthin supplementation may delay age-related loss of fertility and onset of menopause is of significant interest. In a parallel study, either saline or β-cryptoxanthin (5 µg/kg rat/day) was delivered to Wistar albino rats via an osmotic pump from 4 to 7 months of age. All control and β-cryptoxanthin-treated dams were fertile at 7 months of age, with no differences in litter size, sex ratio, or pup viability at the time of mating at 7, 9, 11, and 15 months of age (p ≥.05 for all). As expected, over time there was a pronounced decrease in litter size and serum anti-Müllerian hormone (AMH), but with no significant differences between the two groups at any time point. Overall, there was a positive correlation between litter size and AMH (r = 0.324, p =.012), confirming a link between this serum marker of ovarian reserve status and fertility potential. At 16 months, bilateral oophorectomies were performed at necropsy, before conducting follicle density assessments of ovarian reserve. The total number and stage of follicle development were similar between the β-cryptoxanthin and control groups (13.8 ± 3.2 cf 10.2 ± 4.8, respectively, p >.05). β-cryptoxanthin supplementation for 3 months early in reproductive life was not effective in delaying ovarian senescence or enhancing fertility in rats later in life, contrary to the association suggested by observational studies in humans Refereed/Peer-reviewed

Hypogonadal obese men find it difficult to lose weight. We investigated whether the modification of macronutrient intake can alter testosterone levels independently of the body mass index. Fasted overweight or obese fertile men were asked to consume meals of polyunsaturated fats (PUFA), monounsaturated fats (MUFA), refined carbohydrates (CHO, orange juice, OJ), whey and egg albumin and mixed meals of PUFA and CHO, PUFA and egg albumin, and CHO and egg albumin. Blood was collected at fasting, then hourly for 5 h and analysed to determine the levels of testosterone and other hormones. We found PUFA and MUFA or a mixed meal of PUFA and CHO significantly reduced serum testosterone production to a similar degree over a 5 h period. PUFA decreased serum testosterone levels by 3.2 nmol/L after 1 h compared to baseline (p = 0.023), with this suppression remaining significant up to 5 h postprandially (2.1 nmol/L, p = 0.012). The net overall testosterone levels were reduced by approximately 10 nmol/L &times, h by PUFA, MUFA and PUFA combined with CHO. CHO alone had little effect on testosterone levels, whereas egg albumin was able to increase them (7.4 cf 2.0 nmol/L &times, h). Therefore, for men wishing to optimize their testosterone levels, it may be wise to avoid a high fat intake, drink liquids such as water or OJ or even consider fasting. ANZCTR, Australia, ACTRN12617001034325.

Obesity is associated with implantation failure, miscarriage, low-grade inflammation and a reduction in peripheral immune-suppressive T regulatory (Treg) cells. Therefore, we postulated that this reduction may also extend to the endometrium and cause embryonic loss. In a cohort of 40 infertile women, without implantation failure or recurrent miscarriage, we examined the density of Treg cells, macrophages and natural killer (NK) cells in mid-luteal endometrial biopsies. Significant positive correlations were observed between BMI and endometrial Treg cells and macrophages, but no relationship with NK cells. We postulate that this change may be a positive adaption to minimise adiposity related inflammation.

The aim of this study is to determine Australians’ understanding of the decline in fertility with age, social determinants that influence their decision to start a family and attitudes towards ovarian reserve screening as a tool allowing personalised reproductive life planning. An online survey of 383 childless Australian men and women, aged 18–45 years, was conducted. Both sexes overestimated natural and in vitro fertilization (IVF)-assisted fertility potential with increasing age, with the magnitude of overestimation being more pronounced for men and IVF treatment compared with natural conception. The primary determinants for starting a family were a stable relationship, followed by establishment of career; availability of accessible child care and paid parental leave were considered less important. Finally, the majority of women (74%) would alter their reproductive life planning if they were identified as having low ovarian reserve on screening. Despite increased education, Australians continue to have a poor understanding of age-related decline in natural and IVF-assisted conception, potentially explaining why many delay starting a family. Ovarian reserve screening may help identify individuals at increased risk of premature diminished fertility, giving these women the ability to bring forward their plans for natural conception or undertake fertility preservation (oocyte freezing).

Study question Are Australian fertility clinics adequately addressing unhealthy lifestyle in patients seeking fertility treatment? Summary answer This study has highlighted deficiencies in practices and education around managing patients with unhealthy lifestyle undergoing fertility treatment. What is known already The association between lifestyle and fertility is well documented, with obesity and smoking being of particular concern to fertility and pregnancy outcomes. Guidelines recommend that unhealthy lifestyle is addressed prior to conception, yet anecdotal experience suggests this is not being addressed. Lifestyle modification programmes can be effective in improving pregnancy rates and outcomes, however, recruitment to such programmes can be challenging. Study design size duration A cohort study of Australian fertility clinics' attitudes and practices regarding lifestyle modification to augment reproductive treatment outcomes was conducted between August and October 2015. Participants/materials setting method An online survey was administered to senior fertility nurses, from all registered fertility clinics in Australia. Data were collated and subjected to a univariate data analysis, where frequency tables were produced for each question. A separate qualitative analysis was undertaken of data from open ended questions. Main results and the role of chance Fifty-three out of 85 potential respondents (62.4%), all from different clincis, completed the survey, with almost all acknowledging the importance of addressing unhealthy lifestyle before offering ART treatment. However, most clinics did not offer internal resources to assist with lifestyle modification. Whilst the promotion of healthy lifestyle was recognised as a key component of the role of a fertility nurse, participants did not feel that nurses were best equipped to provide lifestyle modification programmes, owing to a lack of resources and specific skills in this area. Suggested areas for improved practice included prioritising general health prior to offering treatment, and further utilising and upskilling nurses to assist with lifestyle modification programmes. Limitations reasons for caution The survey was completed by only one nurse from each clinic and as such may not be entirely representative of all clinic practices. Wider implications of the findings Fertility clinics are likely to see an increasing number of patients with unhealthy lifestyle, resulting in health concerns such as obesity. The results of this study provide an insight into how unhealthy lifestyle is currently being addressed in fertility clinics and suggested areas that could be targeted for improving practice and outcomes. Study funding/competing interests No conflict of interest to declare. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Trial registration number NA.

Inflammation, both acute and chronic, is associated with testosterone deficiency, raising the possibility of a direct causal link. One potential trigger for inflammation in obese men is the passage of intestinal bacteria into the circulation due to a breakdown in mucosal barrier integrity. Recently, we hypothesized that this endotoxin exposure may cause androgen deficiency in obese men. To test this hypothesis, we analyzed the relationship between serum levels of lipopolysaccharide-binding protein (LBP), an indirect measure of endotoxin exposure, against male reproductive hormones, inflammatory markers (C-reactive protein, IL-1β, IL-6, TNF-α), and adiposity in 75 men. Adiposity was positively correlated with endotoxin exposure (LBP) and inflammation (C-reactive protein, IL-6) and negatively correlated with testosterone. Furthermore, endotoxemia (LBP) was negatively correlated with serum testosterone but positively correlated with IL-6. Multivariate analysis revealed a significant, negative correlation between serum IL-6 and free testosterone. In a second interventional study, low-dose endotoxin challenge in lean men produced a transient inflammatory response that was followed by a decline in serum testosterone, without changes in LH or FSH, providing further evidence that endotoxin-driven inflammation may result in impaired Leydig cell function.

PURPOSE: Mild controlled ovarian hyperstimulation (COH), combined with oocyte retrieval (OR) under local anaesthesia (LA), may provide low-impact IVF. Since a single injection of corifollitrophin alfa (CFA) provides 7 days of COH, we hypothesised that clomiphene-citrate (CC) followed by CFA may provide adequate COH response from one single FSH injection. Therefore, the aim was to assess IVF outcomes after a novel clomiphene citrate/CFA (CC/CFA) protocol, compared to women undergoing standard rFSH COH protocols (good prognosis comparative cohort:GPCC) in a 1:2 matched design. MATERIALS AND METHODS: In this pilot study of 25 patients (ANZCTR id:ACTRN12612000740897, MINIVA:Minimal_Stimulation_in_IVF), we examined the effectiveness of oral clomiphene (100 mg-days 2–6) followed by CFA in a GnRH antagonist protocol producing a single injection COH stimulation regime. All OR were conducted under LA pre-ovarian block. Cycle outcomes were compared to a matched good prognosis comparative cohort (GPCC) undergoing standard rFSH COH. RESULTS: Mild stimulation was achieved with less oocytes being collected compared to the GPCC (6.4 ± 0.7 vs. 10.7 ± 0.9, p

Obesity is associated with both impaired testosterone production and a chronic state of low grade inflammation. Previously it was believed that this inflammation was mediated by a decline in the immunosuppressive action of testosterone. However, more recently an alternative hypothesis (GELDING theory) has suggested that inflammation originating from the passage of intestinal bacteria into the circulation (metabolic endotoxaemia) may actually be the cause of impaired testicular function in obese men. The aim of this study is to investigate if metabolic endotoxaemia, as quantified by serum Lipopolysaccharide Binding Protein (LBP), is associated with impaired testicular endocrine function.A total of 50 men aged between 21 and 50 years (mean 35.1 ± 6.8 years) were assessed for adiposity (BMI, waist circumference and % body fat using bio-impedance), inflammatory status (serum CRP, IL-1β, IL-6, TNFα and LBP) and testicular endocrine function (serum testosterone, estradiol, AMH, LH and FSH). Statistical analysis was performed using Pearson correlation analysis, with log transformation of data where appropriate, and multi-variate regression.Overall increasing adiposity (% body fat) was positively associated with metabolic endotoxaemia (LBP, r = 0.366, p = 0.009) and inflammation (CRP r = 0.531, p 0.001; IL-6 r = 0.463, p = 0.001), while also being negatively correlated with serum testosterone (r = -0.403, p = 0.004). Serum testosterone levels were significantly negatively correlated with inflammation (CRP r = -0.471, p = 0.001; IL-6 r = -0.516, p 0.001) and endotoxaemia (LBP) after adjusting for serum LH levels (p = -0.317, p = 0.03). Furthermore, serum IL-6 was negatively associated with AMH levels (r = -0.324, p = 0.023), with a negative trend between LBP and AMH also approaching significance (r = -0.267, p = 0.064).Obesity and its associated metabolic endotoxaemia helps initiate a pro-inflammatory state characterised by raised serum IL-6 levels, which in turn is correlated with impairment of both Leydig (testosterone) and Sertoli cell function (AMH). These results open up the potential for new treatments of obesity related male hypogonadism that focus on preventing the endotoxaemia associated chronic inflammatory state.L’obésité est associée à la fois à une altération de la production de testostérone et à un état chronique d’inflammation de faible intensité. On pensait auparavant que cette inflammation était induite par une diminution de l’action immunosuppressive de la testostérone. Une hypothèse alternative plus récente (la théorie GELDING) a cependant suggéré que l’inflammation, qui a pour origine le passage de bactéries intestinales dans la circulation (endotoxémie métabolique), pourrait être en fait la cause de l’altération de la fonction endocrine du testicule chez les hommes obèses. Le but de la présente étude était de chercher si l’endotoxémie métabolique, évaluée par la quantification sérique de la Protéine Liant les Lypopolysaccharides (LBP), était associée à une altération de la fonction endocrine du testicule.Un total de 50 hommes âgés de 21 à 50 ans (moyenne 35,1 ± 6,8 ans) ont été évalué pour l’adiposité (IMC, circonférence de la taille, et pourcentage de graisse corporelle par bio-impédance), le statut inflammatoire (CRP, IL-1beta, IL-6, TNFalpha et LBP sériques) et la fonction endocrine du testicule (testostérone, estradiol, AMH, LH et FSH sériques). L’analyse statistique des données a été réalisée par corrélation de Pearson - après transformation logarithmique des données quand nécessaire – et régression multivariée.Une augmentation globale de l’adiposité (% de graisse corporelle) était positivement associée à l’endotoxémie métabolique (LBP, r = 0.366, p = 0.009) et à l’inflammation (CRP r = 0.531, p 0.001; IL-6 r = 0.463, p = 0.001), tout en étant également corrélée négativement à la testostérone sérique (r = −0.403, p = 0.004). Les taux de testostérone sériques étaient, de façon significative, négativement corrélés à l’inflammation (CRP r = −0.471, p = 0.001; IL-6 r = −0.516, p 0.001) et à l’endotoxémie métabolique (LBP) après ajustement sur les taux de LH sériques (p = −0.317, p = 0.03). L’IL-6 sérique était en outre négativement associée aux taux d’AMH (r = −0.324, p = 0.023), avec une tendance négative entre LBP et AMH approchant la signification (r = −0.267, p = 0.064).l’obésité et son endotoxémie métabolique associée favorisent l’initiation d’un état pro-inflammatoire caractérisé par une élévation des taux d’IL-6 sériques, état qui est en retour corrélé à une altération des fonctions des cellules de Leydig (testostérone) et de Sertoli (AMH). Ces résultats ouvrent la possibilité de nouveaux traitements de l’hypogonadisme masculin lié à l’obésité, centrés sur la prévention de l’endotoxémie associée à l’état inflammatoire chronique.L’obésité est associée à la fois à une altération de la production de testostérone et à un état chronique d’inflammation de faible intensité. On pensait auparavant que cette inflammation était induite par une diminution de l’action immunosuppressive de la testostérone. Une hypothèse alternative plus récente (la théorie GELDING) a cependant suggéré que l’inflammation, qui a pour origine le passage de bactéries intestinales dans la circulation (endotoxémie métabolique), pourrait être en fait la cause de l’altération de la fonction endocrine du testicule chez les hommes obèses. Le but de la présente étude était de chercher si l’endotoxémie métabolique, évaluée par la quantification sérique de la Protéine Liant les Lypopolysaccharides (LBP), était associée à une altération de la fonction endocrine du testicule.Un total de 50 hommes âgés de 21 à 50 ans (moyenne 35,1 ± 6,8 ans) ont été évalué pour l’adiposité (IMC, circonférence de la taille, et pourcentage de graisse corporelle par bio-impédance), le statut inflammatoire (CRP, IL-1beta, IL-6, TNFalpha et LBP sériques) et la fonction endocrine du testicule (testostérone, estradiol, AMH, LH et FSH sériques). L’analyse statistique des données a été réalisée par corrélation de Pearson - après transformation logarithmique des données quand nécessaire – et régression multivariée.Une augmentation globale de l’adiposité (% de graisse corporelle) était positivement associée à l’endotoxémie métabolique (LBP, r = 0.366, p = 0.009) et à l’inflammation (CRP r = 0.531, p 0.001; IL-6 r = 0.463, p = 0.001), tout en étant également corrélée négativement à la testostérone sérique (r = −0.403, p = 0.004). Les taux de testostérone sériques étaient, de façon significative, négativement corrélés à l’inflammation (CRP r = −0.471, p = 0.001; IL-6 r = −0.516, p 0.001) et à l’endotoxémie métabolique (LBP) après ajustement sur les taux de LH sériques (p = −0.317, p = 0.03). L’IL-6 sérique était en outre négativement associée aux taux d’AMH (r = −0.324, p = 0.023), avec une tendance négative entre LBP et AMH approchant la signification (r = −0.267, p = 0.064).l’obésité et son endotoxémie métabolique associée favorisent l’initiation d’un état pro-inflammatoire caractérisé par une élévation des taux d’IL-6 sériques, état qui est en retour corrélé à une altération des fonctions des cellules de Leydig (testostérone) et de Sertoli (AMH). Ces résultats ouvrent la possibilité de nouveaux traitements de l’hypogonadisme masculin lié à l’obésité, centrés sur la prévention de l’endotoxémie associée à l’état inflammatoire chronique.

Objective: Endotoxin, also known as lipopolysaccharide (LPS), is a potent immune stimulant. Low levels of endotoxin exposure (metabolic endotoxemia) play a pivotal role in metabolic disorders. However, there is no robust clinical assay to directly quantify metabolic endotoxemia. We aimed to validate the whole blood Endotoxin Activity Assay (EAA™) as a novel, rapid method to quantify low grade metabolic endotoxemia against the well-established lipopolysaccharide binging protein assay (LBP), a robust surrogate marker of endotoxaemia. Methods: 67 women and 47 men aged 21 to 47 years (35.4 ± 5.5 years, 34.5 ±7.2 years respectively) were assessed for adiposity (BMI, waist circumference and % body fat using bio-impedance), endotoxin levels (LBP, EAA™) and inflammatory status (serum CRP, IL-6, IL-8). Results: There was no direct relationship between EAA™ and LBP measures for quantitating metabolic endotoxemia for either women or men (R=0.146, p=0.284; R=0.283 p=0.09 respectively). In women, the traditional indirect marker of endotoxemia LBP correlated significantly with CRP and IL-6, measures of generalised immune activation and inflammation (R=0.664, p

Refereed/Peer-reviewed

The aim of this study is to determine Australians’ understanding of the decline in fertility with age, social determinants that influence their decision to start a family and attitudes towards ovarian reserve screening as a tool allowing personalised reproductive life planning. An online survey of 383 childless Australian men and women, aged 18–45 years, was conducted. Both sexes overestimated natural and in vitro fertilization (IVF)-assisted fertility potential with increasing age, with the magnitude of overestimation being more pronounced for men and IVF treatment compared with natural conception. The primary determinants for starting a family were a stable relationship, followed by establishment of career; availability of accessible child care and paid parental leave were considered less important. Finally, the majority of women (74%) would alter their reproductive life planning if they were identified as having low ovarian reserve on screening. Despite increased education, Australians continue to have a poor understanding of age-related decline in natural and IVF-assisted conception, potentially explaining why many delay starting a family. Ovarian reserve screening may help identify individuals at increased risk of premature diminished fertility, giving these women the ability to bring forward their plans for natural conception or undertake fertility preservation (oocyte freezing).

Study question: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? Summary answer: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. What is known already: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. Study design, size, duration: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2–3 days. A second cohort comprised a crosssectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. Participants/materials, setting, methods: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. Main result and the role of chance: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P , 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P , 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P , 0.002) decreased INSL3 values. Limitations, reasons for caution: These were retrospective studies and the results were obtained fro natural cycles only, with their inherent variability. Wider implications of the findings: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. Refereed/Peer-reviewed

Summary Aim Abnormally functioning immunocompetent cells in the endometrium are thought to be responsible for at least some cases of recurrent reproductive failure [recurrent miscarriage or recurrent in vitro fertilisation (IVF) failure], but their detailed investigation has been hampered by a lack of a standardised protocol of counting such cells in study or control patients. The purpose of this study is to use a standardised protocol for the assessment of immune cells in the endometrial biopsies of a large cohort of women with recurrent reproductive failure and establish relevant reference ranges. Method In a recent study, we reported the presence and distribution of selected immune cells and macrophages in the endometria from 222 women who had a routine endometrial biopsy for investigation of recurrent miscarriage or IVF failure. Since the completion of that study, a further 1767 cases have been examined, using the same assessment parameters of the earlier study. Results This updated analysis of 1989 endometrial biopsies provides reference ranges for CD8 + , CD163 + , CD56 + and CD57 + cells for individual ‘days’ of a normalised menstrual cycle. CD8 + T-cells displayed a modest (50%) increase in numbers in the luteal phase and periglandular aggregation was a useful indicator of a subtle focal endometritis, possibly of infective origin, and generally not identified in H&E sections. A rapid accumulation of CD163 + macrophages occurs in the superficial stroma after day 22 of the cycle, while a significant number of cases displayed single or clustered macrophages within glandular lumens of the superficial endometrium in luteal phase, especially after day 20 of the cycle. The significance of this change is unclear but may relate to a macrophage response to abnormal glandular secretion or to bleeding occurring at the time of ovulation. CD56 + uterine natural killer (uNK) cells show such a dramatic rise in both absolute numbers and percentage of stromal cells from day 22 of the standardised 28 day cycle that this needs to be taken into account in all clinical studies or individual assessments of endometrial biopsies. CD57 + NK cells are seen in small numbers in most cases and cell counts of greater than 10 per mm 2 are regarded as abnormal. Conclusions This large database provides a daily range which is the most accurate survey yet of uNK cell numbers. Co-location of CD8 + T-cells and CD56 + uNK cells in perviascular aggregates has been demonstrated.

What role should legislation or policy play in avoiding the complications of in-vitro fertilization? In this article, we focus on single versus double embryo transfer, and assess three arguments in favour of mandatory single embryo transfer: risks to the mother, risks to resultant children, and costs to society. We highlight significant ethical concerns about each of these. Reproductive autonomy and non-paternalism are strong enough to outweigh the health concerns for the woman. Complications due to non-identity cast doubt on the extent to which children are harmed. Twinning may offer an overall benefit rather than burden to society. Finally, including the future health costs for children (not yet born) in reproductive policy is inconsistent with other decisions. We conclude that mandatory single embryo transfer is not justified and that a number of countries should reconsider their current embryo transfer policy.

Compared with natural conception, IVF is an effective form of fertility treatment associated with higher rates of obstetric complications and poorer neonatal outcomes. While some increased risk is intrinsic to the infertile population requiring treatment, the practice of multiple embryo transfer contributes to these complications and outcomes, especially concerning its role in higher order pregnancies. As a result, several jurisdictions (e.g. Sweden, Belgium, Turkey, and Quebec) have legally mandated elective single-embryo transfer (eSET) for young women. We accept that in very high-risk scenarios (e.g. past history of preterm delivery and poor maternal health), double-embryo transfer (DET) should be prohibited due to unacceptably high risks. However, we argue that mandating eSET for all young women can be considered an unacceptable breach of patient autonomy, especially since DET offers certain women financial and social advantages. We also show that mandated eSET is inconsistent with other practices (e.g. ovulation induction and intrauterine insemination–ovulation induction) that can expose women and their offspring to risks associated with multiple pregnancies. While defending the option of DET for certain women, some recommendations are offered regarding IVF practice (e.g. preimplantation genetic screening and better support of IVF and maternity leave) to incentivise patients to choose eSET.

The early loss of ovarian reserve and subsequent menopause has a major impact on fertility potential and increases the risk of cardiovascular disease, osteoporosis, cognitive decline and mortality later in life. While many studies have reported that lifestyle factors such as diet can influence the age of onset of natural menopause, their results are often contradictory. Therefore, the aim of this study was to examine the influence of diet on the onset of natural menopause using a self-reported food frequency questionnaire in a cohort of 1146 pre-menopausal women followed up for an average of 12.5 years. The primary finding was that the age of natural menopause was positively correlated with dietary intake of the micronutrient -cryptoxanthin (r(2)=0.105, p

Obesity is an increasing public health problem, with two-thirds of the adult population in many Western countries now being either overweight or obese. Male obesity is associated with late onset hypogonadism, a condition characterised by decreased serum testosterone, sperm quality plus diminished fertility and quality of life. In this paper we propose a novel theory underlying the development of obesity related hypogonadism- the GELDING theory (Gut Endotoxin Leading to a Decline IN Gonadal function). Several observational studies have previously reported an association between obesity related hypogonadism (low testosterone) and systemic inflammation. However, for the first time we postulate that the trans-mucosal passage of bacterial lipopolysaccharide (LPS) from the gut lumen into the circulation is a key inflammatory trigger underlying male hypogonadism. Obesity and a high fat/high calorie diet are both reported to result in changes to gut bacteria and intestinal wall permeability, leading to the passage of bacterial endotoxin (lipopolysaccharide- LPS) from within the gut lumen into the circulation (metabolic endotoxaemia), where it initiates systemic inflammation. Endotoxin is known to reduce testosterone production by the testis, both by direct inhibition of Leydig cell steroidogenic pathways and indirectly by reducing pituitary LH drive, thereby also leading to a decline in sperm production. In this paper we also highlight the novel evolutionary benefits of the GELDING theory. Testosterone is known to be a powerful immune-suppressive, decreasing a man's ability to fight infection. Therefore we postulate that the male reproductive axis has evolved the capacity to lower testosterone production during times of infection and resulting endotoxin exposure, decreasing the immunosuppressive influence of testosterone, in turn enhancing the ability to fight infection. While this response is adaptive in times of sepsis, it becomes maladaptive in the setting of "non-infectious" obesity related metabolic endotoxaemia.L’obésité est un problème de santé publique en expansion ; les deux-tiers de la population adulte de nombreux Pays de l’Ouest sont actuellement soit en surpoids, soit obèses. L’obésité masculine est associée à l’hypogonadisme de survenue tardive, une situation caractérisée par un taux abaissé de testostérone, une qualité spermatique réduite ainsi qu’une diminution de la fertilité et de la qualité de vie. Dans le présent article, nous proposons une nouvelle théorie sous-tendant le développement de l’hypogonadisme lié à l’obésité – la théorie GELDING (Gut Endotoxin Leading to a Decline IN Gonadal function ; Endotoxines intestinales donnant lieu à un déclin de la fonction gonadique).Plusieurs études observationnelles ont précédemment rapporté une association entre hypogonadisme (testostérone basse) lié à l’obésité et inflammation systémique. Toutefois, nous postulons pour la première fois que le passage trans-muqueuse de lypopolysaccharides bactériens (LPS) de la lumière intestinale dans la circulation constitue un élément inflammatoire clé déclencheur de l’hypogonadisme masculin. L’obésité et une alimentation riche en graisse/riche en calories sont toutes deux signalées pour induire des modifications des bactéries intestinales et de la perméabilité de la paroi intestinale, conduisant au passage d’endotoxines bactériennes (lipopolysaccharide- LPS) de l’intérieur de la lumière intestinale dans la circulation (endotoxémie métabolique) où elles initient une inflammation systémique. Les endotoxines sont connues pour réduire la production de testostérone par les testicules, à la fois par inhibition directe des voies de la stéroïdogenèse des cellules de Leydig et indirectement par la réduction du pic de LH hypophysaire, ce qui conduit aussi à une réduction de la production de spermatozoïdes.Dans le présent article, nous avons aussi mis en relief nouveaux bénéfices évolutionnaires de la théorie GELDING. La testostérone est connue pour être un puissant immunosuppresseur qui diminue la capacité d’un homme à combattre l’infection. Par conséquent, nous postulons que l’axe reproducteur masculin a élaboré la capacité à diminuer la production de testostérone pendant les périodes d’infection et d’exposition aux endotoxines qui en résulte, ce qui réduit ainsi l’influence immunosuppressive de la testostérone, et en retour augmente la capacité de combattre l’infection. Alors que cette réponse est adaptée en période de sepsis, elle devient inadaptée dans le cadre de l’endotoxémie métabolique ‘non infectieuse’ liée à l’obésité.

Obesity is known to be associated with an increased risk of miscarriage after natural and assisted conception. Although most sporadic miscarriages are caused by genetic abnormalities, it is presently uncertain if genetics is also the underlying mechanism leading to increased pregnancy loss seen in obese women. Karyotyping of the products of conception suggests a reduced rate of fetal aneuploidy in miscarriages from obese compared with lean individuals. Karyotype analysis, however, is prone to false negative results because of inadvertent culture of maternal rather than fetal tissue. Therefore, to better analyse the effect of the genetic status on obesity-related miscarriage, we retrospectively analysed the outcomes 125 consecutive cryopreserved embryo transfer cycles resulting in a pregnancy after screening for genetic normality using comparative genomic hydridization. Lean individuals (body mass index 18.5–24.9 kg/m2) had a significantly lower rate of miscarriages (14.2%) than overweight (29.1%) or obese (41.9%) women (P = 0.001); this relationship remained significant (P = 0.023) even after adjusting for relevant confounders, e.g. maternal age, cause of infertility, number of previous IVF cycles, type of frozen embryo transfer cycle or past obstetric history. These results support a non-genetic cause for obesity-related miscarriage. Refereed/Peer-reviewed

Study question Do seminal plasma transforming growth factor-β (TGFB) cytokines vary within individuals over time, and does this relate to sperm parameters, age or prior abstinence? Summary answer Activin A and follistatin, and to a lesser extent TGFB1, TGFB2 and TGFB3, vary within individuals over time, in association with duration of abstinence. What is already known Seminal plasma TGFB cytokines can influence sperm function and reproductive success through interactions with the female reproductive tract after coitus. Over time, individual sperm parameters fluctuate considerably. Whether seminal fluid TGFB cytokines vary similarly, and the determinants of any variance, is unknown. Study design, size, duration Between two and seven semen samples were collected from each of 14 fertile donors at 6-10 week intervals over the course of 12 months, then seminal plasma cytokines and sperm parameters were measured. Participants/materials, setting and method The concentrations and total amounts per ejaculate of TGFB1, TGFB2, TGFB3, activin A and follistatin were determined using commercial assays. Sperm parameters were assessed according to WHO IV standards. Mixed model analysis was utilised to determine the relative contribution of between- and within-individual factors to the variance. Relationships between cytokines and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. Main results and the role of chance Within-individual variability contributed to the total variance for all cytokines and sperm parameters, and was a stronger determinant than between-individual variability for activin A and follistatin as well as for total sperm concentration and sperm motility. Positive correlations between each of the three TGFB isoforms, and activin and follistatin, suggest co-regulation of synthesis. Duration of abstinence influenced total content of TGFB1, TGFB2, activin A and follistatin. TGFB1 correlated inversely with age. Limitations, reasons for caution A limited number of donors from a single clinic were investigated. Clinical information on BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalised to larger populations and different ethnicities. Wider implications of the findings These data reveal substantial variation over time in seminal fluid cytokines and indicate that repeated analyses are required to gain precise representative data on an individual's status. Within-individual variation in seminal fluid components should be taken into account when investigating seminal fluid cytokines. Study funding/competing interests This study was supported by grants from the National Health and Medical Research Council of Australia, ID453556 and APP1041332. The authors have no competing interests to disclose.

STUDY QUESTION; Does the presence of ultrasound diagnosed adenomyosis interfere with successful implantation in patients undergoing IVF treatment with GnRH antagonist ovarian stimulation? BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: Given the retrospective nature of this study, there is risk of bias. This risk was minimized by having subjective variables such as embryo quality assessed by individuals not involved in the study, while strictly applying the pre-determined inclusion/exclusion criteria to all study participants. Furthermore, it is acknowledged that ultrasound is not a perfect test for the diagnosis of adenomyosis and, therefore, may underestimate the incidence of adenomyosis by misclassifying some patients with mild adenomyosis as not affected. DESIGN: This is a retrospective cohort study of 748 patients who, between April 2010 and March 2012, underwent a screening transvaginal ultrasound to identify possible pelvic pathology before commencing their IVF treatment. From this screening group, 213 patients were eligible to be included in the study as they had no obvious underlying uterine or embryonic factors that could have interfered with successful implantation (aged ≤39 years, good quality Day 4/5 embryo for single-embryo transfer, no uterine fibroids/hydrosalpinx or endometrial polyps). MAIN RESULTS AND THE ROLE OF CHANCE: The adenomyosis group had a viable clinical pregnancy rate of 23.6% compared with 44.6% in the non-adenomyosis group (P =0.017). However, the median maternal age and duration of infertility of the adenomyosis group was 2 years older and 4 months greater, respectively, than that of the non-adenomyosis group. A logistic regression analysis was performed to account for these differences between the two groups, with the adjusted results still showing a statistically significant decline in viable pregnancy rate in the adenomyosis group (OR = 0.408, CI = 0.181-0.922, P =0.031 when adjusting for maternal age; OR = 0.417, CI = 0.175-0.989, P =0.047 when adjusting for duration of infertility) PARTICIPANTS AND SETTING: There were 213 patients in a private IVF unit eligible to be included in the study, with 38 patients (17.84%) having ultrasound diagnosed adenomyosis and 175 patients having no adenomyosis on the scan. Only the first treatment cycle for each patient was included. SUMMARY ANSWER: The presence of ultrasound diagnosed adenomyosis was associated with a significant reduction in successful implantation of good quality embryos in patients undergoing GnRH antagonist stimulation for IVF treatment (viable clinical pregnancy rate 23.6% versus 44.6%, P= 0.017). WHAT IS KNOWN AND WHAT THIS PAPER ADDS: There is currently no consensus regarding the impact of adenomyosis on implantation potential. Although some studies have identified alterations in the endometrial milieu in adenomyosis patients that may impact implantation, several papers have reported no associated reproductive deficit. However, these pregnancy outcome studies have primarily investigated patients undergoing long down-regulation IVF protocols, where low levels of serum estrogen (before commencing the ovarian stimulation) may inactivate the adenomyosis and potentially negate its effect on implantation. Given that the majority of fertility clinics are now moving towards the more 'patient-friendly' antagonist protocol, where patients are not placed in a hypo-estrogen state before commencing ovarian stimulation, the question of whether adenomyosis has an impact on IVF success rates in GnRH antagonist-stimulated IVF treatment needs to be examined. WIDER IMPLICATIONS OF THE FINDINGSThe results of this study should be representative of outcomes for any patient undergoing a GnRH antagonist ovarian stimulation cycle for IVF since standard IVF treatment protocols were used. Refereed/Peer-reviewed

In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. In this study, we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner’s seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the periovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. A substantial influx of CD45+ cells mainly comprising CD14+ macrophages and CD1a+ dendritic cells expressing CD11a and MHC class II was evident in both the stratified epithelium and deeper stromal tissue after coitus. CD3+CD8+CD45RO+ T cells were also abundant and increased after coitus. Leukocyte recruitment did not occur without coitus or with condom-protected coitus. An accompanying increase in CSF2, IL6, IL8, and IL1A expression was detected by quantitative RT-PCR, and microarray analysis showed genes linked with inflammation, immune response, and related pathways are induced by seminal fluid in cervical tissues. We conclude that seminal fluid introduced at intercourse elicits expression of proinflammatory cytokines and chemokines, and a robust recruitment of macrophages, dendritic cells, and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens and potentially, susceptibility to cervical metaplasia.

Adenomyosis, a condition usually associated with multiparity, is not generally seen as a cause of infertility. However, recent studies have reported a reduction in IVF implantation rates and a link with miscarriage, suggesting that adenomyosis may interfere with successful implantation. To investigate this hypothesis, the clinical records and laboratory results, which routinely include immunohistochemical examination of a late luteal phase endometrial biopsy for leukocytes, were retrospectively reviewed for 64 women with implantation failure and who previously had been screened for the presence of adenomyosis by pelvic MRI. The presence of either diffuse or "adenomyoma" type of adenomyosis was associated with a marked increase (p=0.004) in the density of macrophages and natural killer cells in the endometrial stroma, compared to those women with mild focal adenomyosis or no disease. These findings point to an immunological mechanism by which adenomyosis might interfere with successful embryo implantation.

Recurrent miscarriage affects approximately 1% of the population and in half of these cases no cause is found. Abnormally functioning immunocompetent cells, including natural killer (NK) cells, in the endometrium, are thought to be responsible for many such cases and treatment trials including oral prednisolone and intravenous immunoglobulins are now underway. Despite these encouraging developments, there is neither adequate standardization of counting uterine NK cells nor consensus as to what constitutes an abnormal level. To address this issue, immunohistochemistry was used to examine the presence and distribution of selected immune cells and macrophages in the endometrium from 222 women who had a routine endometrial biopsy for investigation of recurrent miscarriage or IVF failure, at various stages of the menstrual cycle, and accessioned prospectively over a 7-month period. Biopsies were examined by H+E and immunostained for CD8(+) T-cells, CD163(+) macrophages, CD56(+) NK cells, and CD57(+) cells. Cell numbers (expressed as immunopositive cells per mm(2)) were determined in the stroma of the functional layer of endometrium and the relative concentrations of some cell types (CD163(+) macrophages, CD56(+) NK cells) were expressed as a percentage of all stromal cells. Routine H+E sections revealed 12 patients with focal "endometritis" without plasma cells. CD8(+) T-cells showed focal perivascular aggregates in most instances, and non-random but scattered cells in all cases, with a twofold increase in the luteal phase. CD163(+) cells were distributed evenly throughout the superficial endometrial stroma and also present as single or clustered macrophages within the lumens of superficial glands, mostly in the luteal phase. CD56(+) NK cells showed "diffuse" but variable distribution throughout the functional layer and perivascular aggregates of various sizes in two thirds of cases. Raw cell counts were low and relatively stable in the proliferative phase, but increased somewhat during the first half of the secretory phase, while in the second half of secretory phase they increased six to tenfold. Percentage counts rose from approximately 5% of stromal cells in the early part of the secretory phase of the cycle to over 35% in premenstrual endometrium. CD57(+) cells were present in very low numbers in most cases. The study illustrates the complexity and variability of immune cell infiltration of endometrium. We stress the need for strict counting protocols and attention to histological criteria if any immunological perturbations potentially responsible for recurrent reproductive failure are to be identified. Reference ranges for individual cell types are only valid for individual "days" of a normalized menstrual cycle.