The metabolic health of young men conceived using intracytoplasmic sperm injection.

STUDY QUESTION: Is the metabolic health of men conceived using ICSI different to that of IVF and spontaneously conceived (SC) men? SUMMARY ANSWER: ICSI-conceived men aged 18-24 years, compared with SC controls, showed differences in some metabolic parameters including higher resting diastolic blood pressure (BP) and homeostasis model assessment for insulin resistance (HOMA-IR) scores, although the metabolic parameters of ICSI- and IVF-conceived singleton men were more comparable. WHAT IS KNOWN ALREADY: Some studies suggest that IVF-conceived offspring may have poorer cardiovascular and metabolic profiles than SC children. Few studies have examined the metabolic health of ICSI-conceived offspring. STUDY DESIGN, SIZE, DURATION: This cohort study compared the metabolic health of ICSI-conceived men to IVF-conceived and SC controls who were derived from prior cohorts. Participants included 121 ICSI-conceived men (including 100 singletons), 74 IVF-conceived controls (all singletons) and 688 SC controls (including 662 singletons). PARTICIPANTS/MATERIALS, SETTING, METHODS: Resting systolic and diastolic BP (measured using an automated sphygmomanometer), height, weight, BMI, body surface area and fasting serum metabolic markers including fasting insulin, glucose, total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol, triglycerides, highly sensitive C-reactive protein (hsCRP) and HOMA-IR were compared between groups. Data were analysed using multivariable linear regression adjusted for various covariates including age and education level. MAIN RESULTS AND THE ROLE OF CHANCE: After adjusting for covariates, compared to 688 SC controls, 121 ICSI-conceived men had higher diastolic BP (ß 4.9, 95% CI 1.1-8.7), lower fasting glucose (ß -0.7, 95% CI -0.9 to -0.5), higher fasting insulin (ratio 2.2, 95% CI 1.6-3.0), higher HOMA-IR (ratio 1.9, 95% CI 1.4-2.6), higher HDLC (ß 0.2, 95% CI 0.07-0.3) and lower hsCRP (ratio 0.4, 95% CI 0.2-0.7) levels. Compared to 74 IVF-conceived singletons, only glucose differed in the ICSI-conceived singleton men (ß -0.4, 95% CI -0.7 to -0.1). No differences were seen in the paternal infertility subgroups. LIMITATIONS, REASONS FOR CAUTION: The recruitment rate of ICSI-conceived men in this study was low and potential for recruitment bias exists. The ICSI-conceived men, the IVF-conceived men and SC controls were from different cohorts with different birth years and different geographical locations. Assessment of study groups and controls was not contemporaneous, and the measurements differed for some outcomes (BP, insulin, glucose, lipids and hsCRP). WIDER IMPLICATIONS OF THE FINDINGS: These observations require confirmation in a larger study with a focus on potential mechanisms. Further efforts to identify whether health differences are due to parental characteristics and/or factors related to the ICSI procedure are also necessary. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by an Australian National Health and Medical Research Council Partnership Grant (NHMRC APP1140706) and was partially funded by the Monash IVF Research and Education Foundation. S.R.C. was supported through an Australian Government Research Training Program Scholarship. R.J.H. is supported by an NHMRC project grant (634457), and J.H. and R.I.M. have been supported by the NHMRC as Senior and Principal Research Fellows respectively (J.H. fellowship number: 1021252; R.I.M. fellowship number: 1022327). L.R. is a minority shareholder and the Group Medical Director for Monash IVF Group, and reports personal fees from Monash IVF Group and Ferring Australia, honoraria from Ferring Australia and travel fees from Merck Serono and MSD and Guerbet; R.J.H. is the Medical Director of Fertility Specialists of Western Australia and has equity in Western IVF; R.I.M. is a consultant for and shareholder of Monash IVF Group and S.R.C. reports personal fees from Besins Healthcare and nonfinancial support from Merck outside of the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Exome sequencing reveals variants in known and novel candidate genes for severe sperm motility disorders.

STUDY QUESTION: What are the causative genetic variants in patients with male infertility due to severe sperm motility disorders? SUMMARY ANSWER: We identified high confidence disease-causing variants in multiple genes previously associated with severe sperm motility disorders in 10 out of 21 patients (48%) and variants in novel candidate genes in seven additional patients (33%). WHAT IS KNOWN ALREADY: Severe sperm motility disorders are a form of male infertility characterised by immotile sperm often in combination with a spectrum of structural abnormalities of the sperm flagellum that do not affect viability. Currently, depending on the clinical sub-categorisation, up to 50% of causality in patients with severe sperm motility disorders can be explained by pathogenic variants in at least 22 genes. STUDY DESIGN, SIZE, DURATION: We performed exome sequencing in 21 patients with severe sperm motility disorders from two different clinics. PARTICIPANTS/MATERIALS, SETTING, METHOD: Two groups of infertile men, one from Argentina (n = 9) and one from Australia (n = 12), with clinically defined severe sperm motility disorders (motility <5%) and normal morphology values of 0-4%, were included. All patients in the Argentine cohort were diagnosed with DFS-MMAF, based on light and transmission electron microscopy. Sperm ultrastructural information was not available for the Australian cohort. Exome sequencing was performed in all 21 patients and variants with an allele frequency of <1% in the gnomAD population were prioritised and interpreted. MAIN RESULTS AND ROLE OF CHANCE: In 10 of 21 patients (48%), we identified pathogenic variants in known sperm assembly genes: CFAP43 (3 patients); CFAP44 (2 patients), CFAP58 (1 patient), QRICH2 (2 patients), DNAH1 (1 patient) and DNAH6 (1 patient). The diagnostic rate did not differ markedly between the Argentinian and the Australian cohort (55% and 42%, respectively). Furthermore, we identified patients with variants in the novel human candidate sperm motility genes: DNAH12, DRC1, MDC1, PACRG, SSPL2C and TPTE2. One patient presented with variants in four candidate genes and it remains unclear which variants were responsible for the severe sperm motility defect in this patient. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we described patients with either a homozygous or two heterozygous candidate pathogenic variants in genes linked to sperm motility disorders. Due to unavailability of parental DNA, we have not assessed the frequency of de novo or maternally inherited dominant variants and could not determine the parental origin of the mutations to establish in all cases that the mutations are present on both alleles. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirm the likely causal role of variants in six known genes for sperm motility and we demonstrate that exome sequencing is an effective method to diagnose patients with severe sperm motility disorders (10/21 diagnosed; 48%). Furthermore, our analysis revealed six novel candidate genes for severe sperm motility disorders. Genome-wide sequencing of additional patient cohorts and re-analysis of exome data of currently unsolved cases may reveal additional variants in these novel candidate genes. STUDY FUNDING/COMPETING INTEREST(S): This project was supported in part by funding from the Australian National Health and Medical Research Council (APP1120356) to M.K.O.B., J.A.V. and R.I.M.L., The Netherlands Organisation for Scientific Research (918-15-667) to J.A.V., the Royal Society and Wolfson Foundation (WM160091) to J.A.V., as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V. and Grants from the National Research Council of Argentina (PIP 0900 and 4584) and ANPCyT (PICT 9591) to H.E.C. and a UUKi Rutherford Fund Fellowship awarded to B.J.H.

Health and fertility of ICSI-conceived young men: study protocol.

STUDY QUESTIONS: What are the long-term health and reproductive outcomes for young men conceived using ICSI whose fathers had spermatogenic failure (STF)? Are there epigenetic consequences of ICSI conception? WHAT IS KNOWN ALREADY: Currently, little is known about the health of ICSI-conceived adults, and in particular the health and reproductive potential of ICSI-conceived men whose fathers had STF. Only one group to date has assessed semen parameters and reproductive hormones in ICSI-conceived men and suggested higher rates of impaired semen quality compared to spontaneously conceived (SC) peers. Metabolic parameters in this same cohort of men were mostly comparable. No study has yet evaluated other aspects of adult health. STUDY DESIGN SIZE DURATION: This cohort study aims to evaluate the general health and development (aim 1), fertility and metabolic parameters (aim 2) and epigenetic signatures (aim 3) of ICSI-conceived sons whose fathers had STF (ICSI study group). There are three age-matched control groups: ICSI-conceived sons whose fathers had obstructive azoospermia (OAZ) and who will be recruited in this study, as well as IVF sons and SC sons, recruited from other studies. Of 1112 ICSI parents including fathers with STF and OAZ, 78% (n = 867) of mothers and 74% (n = 823) of fathers were traced and contacted. Recruitment of ICSI sons started in March 2017 and will finish in July 2020. Based on preliminary participation rates, we estimate the following sample size will be achieved for the ICSI study group: mothers n = 275, fathers n = 225, sons n = 115. Per aim, the sample sizes of OAZ-ICSI (estimated), IVF and SC controls are: Aim 1-OAZ-ICSI: 28 (maternal surveys)/12 (son surveys), IVF: 352 (maternal surveys)/244 (son surveys), SC: 428 (maternal surveys)/255 (son surveys); Aim 2-OAZ-ICSI: 12, IVF: 72 (metabolic data), SC: 391 (metabolic data)/365 (reproductive data); Aim 3-OAZ-ICSI: 12, IVF: 71, SC: 292. PARTICIPANTS/MATERIALS SETTING METHODS: Eligible parents are those who underwent ICSI at one of two major infertility treatment centres in Victoria, Australia and gave birth to one or more males between January 1994 and January 2000. Eligible sons are those aged 18 years or older, whose fathers had STF or OAZ, and whose parents allow researchers to approach sons. IVF and SC controls are age-matched men derived from previous studies, some from the same source population. Participating ICSI parents and sons complete a questionnaire, the latter also undergoing a clinical assessment. Outcome measures include validated survey questions, physical examination (testicular volumes, BMI and resting blood pressure), reproductive hormones (testosterone, sex hormone-binding globulin, FSH, LH), serum metabolic parameters (fasting glucose, insulin, lipid profile, highly sensitive C-reactive protein) and semen analysis. For epigenetic and future genetic analyses, ICSI sons provide specimens of blood, saliva, sperm and seminal fluid while their parents provide a saliva sample. The primary outcomes of interest are the number of mother-reported hospitalisations of the son; son-reported quality of life; prevalence of moderate-severe oligozoospermia (sperm concentration <5 million/ml) and DNA methylation profile. For each outcome, differences between the ICSI study group and each control group will be investigated using multivariable linear and logistic regression for continuous and binary outcomes, respectively. Results will be presented as adjusted odds ratios and 95% CIs. STUDY FUNDING/COMPETING INTERESTS: This study is funded by an Australian National Health and Medical Research Council Partnership Grant (NHMRC APP1140706) and was partially funded by the Monash IVF Research and Education Foundation. L.R. is a minority shareholder and the Group Medical Director for Monash IVF Group, and reports personal fees from Monash IVF group and Ferring Australia, honoraria from Ferring Australia, and travel fees from Merck Serono, MSD and Guerbet; R.J.H. is the Medical Director of Fertility Specialists of Western Australia and has equity in Western IVF; R.I.M. is a consultant for and a shareholder of Monash IVF Group and S.R.C. reports personal fees from Besins Healthcare and non-financial support from Merck outside of the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable. TRIAL REGISTRATION DATE: Not applicable. DATE OF FIRST PATIENT'S ENROLMENT: Not applicable.

Supportive oncofertility care, psychological health and reproductive concerns: a qualitative study.

PURPOSE: Impaired fertility in cancer patients and survivors of reproductive age (15-45 years) may lead to psychological distress and poor mental health outcomes, and may negatively impact quality of life. Limited research has focused on the fertility experiences of those who have had access to supportive oncofertility care. This study aims to explore the fertility-care experiences and reproductive concerns of reproductive age cancer patients at the time of their cancer diagnosis who have had access to oncofertility care. METHODS: The qualitative data from a larger mixed method study is presented, comprising 30 semi-structured telephone interviews with newly diagnosed cancer patients across Australia and New Zealand, undertaken between April 2016 and April 2018. RESULTS: Interviews were undertaken with 9 male patients and 21 female patients aged between 15 and 44 years. All patients recalled a discussion about fertility and majority underwent some form of fertility preservation. Thematic analysis identified five main themes: (i) satisfaction with oncofertility care, (ii) a need for individualised treatment and support, (iii) desire for parenthood, (iv) fertility treatment can be challenging, and (v) fertility preservation provides a safety net for the future. CONCLUSIONS: Participants who access supportive oncofertility care report low emotional impact of threatened future infertility at the time of cancer diagnosis. These results suggest that such services may assist in lowering the emotional burden of potential infertility in survivors. Long-term research is needed to assess the longitudinal benefits for different models of care.

Germ cell arrest associated with aSETX mutation in ataxia oculomotor apraxia type 2.

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar atrophy, peripheral neuropathy and oculomotor apraxia. It is caused by mutations in the SETX gene that encodes senataxin, a ubiquitously expressed protein that mediates processes, including transcription, transcription termination, DNA repair, RNA processing, DNA-RNA hybrid (R-loop) elimination and telomere stability. In mice, senataxin is essential for male germ cell development and fertility through its role in meiotic recombination and sex chromosome inactivation. AOA2 is associated with hypogonadism in women, but there are no reports of hypogonadism or infertility in men. We describe the first case of human male infertility caused by germ cell arrest in a man with AOA2. Our patient has a homozygous mutation in the SETX gene (NC_000009.11:g.135158775dup), which results in a frameshift and premature protein termination (NM_015046.6:c.6422dup, p.[Ser2142Glufs*23]). In accordance with the murine phenotype, testis histology revealed disrupted seminiferous tubules with spermatogonia and primary spermatocytes, but absent spermatids. Collectively, these data support an essential role of senataxin in human spermatogenesis, and provide a compelling case that men with AOA2 should be counselled at diagnosis about the possibility of infertility.

Long-term follow-up of ICSI-conceived offspring compared with spontaneously conceived offspring: a systematic review of health outcomes beyond the neonatal period.

BACKGROUND: A significant increase in the use of intracytoplasmic sperm injection (ICSI) since its introduction in 1992 has been observed worldwide, including beyond its original intended use for severe male factor infertility. Concerns regarding ICSI include the effects of poor quality spermatozoa on offspring health and future fertility, and of the technique itself. The health and development of ICSI-conceived children beyond early infancy have not been comprehensively assessed. OBJECTIVE: A systematic review of health outcomes of ICSI-conceived offspring beyond the neonatal period compared to spontaneously conceived (SC) offspring. DESIGN: PubMed, OVID Medline/Embase, InformIT, Web of Science, and ProQuest databases were searched for studies reporting on health outcomes in ICSI-conceived offspring beyond 28 days after birth. MAIN OUTCOMES MEASURE(S): Physical and psychosocial health. RESULTS: The search strategy yielded 2826 articles. Of these, 2580 were not relevant or did not meet inclusion criteria and 138 were duplicates. One hundred and eight full-text papers were evaluated further, and 48 satisfied the inclusion criteria. Most studies reported on neurodevelopment during early infancy and childhood with reassuring results. Growth, vision, and hearing of ICSI and SC offspring also appear comparable, although important differences in general physical health, and particularly metabolic and reproductive health have been described, including recently poorer semen quality among ICSI-conceived young adult men compared to SC peers. CONCLUSION: Whilst neurodevelopment, growth, vision, and hearing appear similar between ICSI and SC children, evidence suggests differences in general physical health, and metabolic and reproductive endpoints. The clinical significance of many findings, however, remains unclear, and further prospective, large, and good quality studies with a focus on all these health outcomes in ICSI-conceived young adults are required.

Survey of Reproductive Experiences and Outcomes of Cancer Survivors Who Stored Reproductive Material Before Treatment.

STUDY QUESTION: What are the reproductive experiences and outcomes of people who store reproductive material before cancer treatment? SUMMARY ANSWER: Of respondents who had tried to achieve pregnancy since completing cancer treatment almost all had succeeded, in most cases through natural conception. WHAT IS KNOWN ALREADY: People of reproductive age who are diagnosed with cancer can cryopreserve reproductive material to guard against the adverse effects on fertility of gonadotoxic treatment. Little is known about the reproductive outcomes of people who undergo fertility preservation before cancer treatment. STUDY DESIGN, SIZE, DURATION: Cross-sectional survey. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women and men who had stored reproductive material before cancer treatment at two private and one public fertility clinics up to June 2014 and were at least 18 years old at the time were identified from medical records and invited to complete an anonymous questionnaire about their reproductive experiences. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 870 potential respondents 302 (171 female and 131 male) returned completed questionnaires yielding a response rate of 34.5% (39.5% and 29.7% for female and male respondents, respectively). Current age was similar for women and men (37.2 years) but men had been diagnosed with cancer significantly earlier in life than women (28.2 versus 30.3 years, P = 0.03). Almost two-thirds of respondents wished to have a child or another child in the future, some of whom knew that they were unable to. One in ten respondents was a parent before the cancer diagnosis and around one-third had had a child since diagnosis or was pregnant (or a partner in pregnancy) at the time of the survey. Of those who had tried to conceive since completing cancer treatment (N = 119) 84% (79% of women and 90% of men) had had a child or were pregnant (or a partner in pregnancy). Most of the pregnancies since the diagnosis of cancer occurred after natural conception (58/100, 58%). Of the 22 women (13% of all women) and 35 men (27% of all men) who had used their stored reproductive material four women (18%) and 28 men (80%) had had a child or were pregnant or a partner in pregnancy at the time of completing the survey. The most commonly stated reason for not using the stored material was not being ready to try for a baby. LIMITATIONS, REASON FOR CAUTION: The relatively low response rate, particularly among men, means that participation bias may have influenced the findings. As type of cancer was self-reported and we did not ask questions about respondents' cancer treatments, it is not possible to link reproductive outcomes to type of cancer or cancer treatment. Also, there is no way of comparing the sample with the populations they were drawn from as data on reproductive outcomes of people who store reproductive material before cancer treatment are not collected routinely. This might have led to over- or underestimates of the reproductive experiences and outcomes reported in this paper. WIDER IMPLICATIONS OF THE FINDINGS: The findings add to the limited evidence about the reproductive outcomes of this growing group of people and can be used to inform the advice given to those contemplating fertility preservation in the context of cancer. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the National Health and Medical Research Council (APP1042347). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Long-term follow-up of intra-cytoplasmic sperm injection-conceived offspring compared with in vitro fertilization-conceived offspring: a systematic review of health outcomes beyond the neonatal period.

The use of intra-cytoplasmic sperm injection (ICSI) has increased significantly worldwide, often chosen instead of in vitro fertilization (IVF), yet long-term health outcomes are unknown and health differences between ICSI and IVF conceptions have not been comprehensively assessed. A systematic review of health outcomes of ICSI-conceived offspring beyond the neonatal period compared to IVF-conceived offspring was carried out. PubMed, OVID Medline/Embase, Informit, Web of Science and Proquest databases were searched on 9 November 2016 for studies reporting on health outcomes in ICSI-conceived offspring beyond 28 days after birth. Physical and psychosocial health were the main outcome measures. The search strategy yielded 2781 articles; 2539 were not relevant or did not meet inclusion criteria and 137 were duplicates. One hundred and five full-text papers were evaluated further and 34 satisfied the inclusion criteria. Studies comparing ICSI- and IVF-conceived children suggest their neurodevelopment is comparable. Growth and aspects of physical health are also similar; however, studies are few and limited to childhood. ICSI-conceived children may be at increased risk of autism and intellectual impairment. No difference in risk of childhood cancer was reported in one study. Whilst the neurodevelopment of ICSI-conceived children appears comparable to those of IVF conception, data relating to neurodevelopmental disorders, growth, physical health and childhood cancer are inconclusive. Further research into health outcomes in adolescence and adulthood is required before conclusions can be drawn about the long-term safety of ICSI compared to IVF. Until then, ICSI might be better reserved for its original intended use, male-factor infertility.

Efficient and accurate methods for solving the time-dependent spin-1 Gross-Pitaevskii equation.

We develop a numerical method for solving the spin-1 Gross-Pitaevskii equation. The basis of our work is a two-way splitting of the spin-1 evolution equation that leads to two exactly solvable flows. We use this to implement a second-order and a fourth-order symplectic integration method. These are the first fully symplectic methods for evolving spin-1 condensates. We develop two nontrivial numerical tests to compare our methods against two other approaches.

Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

STUDY QUESTION: Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER: Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY: Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION: BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), ß-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and ß-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE: Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, ß-catenin and ß-actin did not differ, compared with control. LIMITATIONS, REASONS FOR CAUTION: Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. WIDER IMPLICATIONS OF THE FINDINGS: These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. STUDY FUNDING/COMPETING INTERESTS: National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Testicular function in a birth cohort of young men.

STUDY QUESTION: By investigating a birth cohort with a high ongoing participation rate to derive an unbiased population, what are the parameters and influences upon testicular function for a population not selected with regard to fertility? SUMMARY ANSWER: While varicocele, cryptorchidism and obesity may impact on human testicular function, most common drug exposures and the presence of epididymal cysts appear to have no or minimal adverse impact. WHAT IS KNOWN ALREADY: The majority of previous attempts to develop valid reference populations for spermatogenesis have relied on potentially biased sources such as recruits from infertility clinics, self-selected volunteer sperm donors for research or artificial insemination or once-fertile men seeking vasectomy. It is well known that studies requiring semen analysis have low recruitment rates which consequently question their validity. However, there has been some concern that a surprisingly high proportion of young men may have semen variables that do not meet all the WHO reference range criteria for fertile men, with some studies reporting that up to one half of participants have not meet the reference range for fertile men. Reported median sperm concentrations have ranged from 40 to 60 million sperm/ml. STUDY DESIGN, SIZE AND DURATION: The Western Australian Pregnancy Cohort (Raine) was established in 1989. At 20-22 years of age, members of the cohort were contacted to attend for a general follow-up, with 753 participating out of the 913 contactable men. Of these, 423 men (56% of participants in the 20-22 years cohort study, 46% of contactable men) participated in a testicular function study. Of the 423 men, 404 had a testicular ultrasound, 365 provided at least one semen sample, 287 provided a second semen sample and 384 provided a blood sample. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular ultrasound examinations were performed at King Edward Memorial Hospital, Subiaco, Perth, for testicular volume and presence of epididymal cysts and varicoceles. Semen samples were provided and analysed by standard semen assessment and a sperm chromatin structural assay (SCSA) at Fertility Specialists of Western Australia, Claremont, Perth. Serum blood samples were provided at the University of Western Australia, Crawley, Perth and were analysed for serum luteinizing hormone (LH), follicular stimulating hormone (FSH), inhibin B, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), estradiol, estrone and the primary metabolites of DHT: 5α-androstane-3α,17ß-diol (3α-diol) and 5-α androstane-3-ß-17-beta-diol (3ß-diol). Serum steroids were measured by liquid chromatography, mass spectrometry and LH, FSH and inhibin B were measured by ELISA assays. MAIN RESULTS AND THE ROLE OF CHANCE: Cryptorchidism was associated with a significant reduction in testicular (P = 0.047) and semen (P = 0.027) volume, sperm concentration (P = 0.007) and sperm output (P = 0.003). Varicocele was associated with smaller testis volume (P < 0.001), lower sperm concentration (P = 0.012) and total sperm output (P = 0.030) and lower serum inhibin B levels (P = 0.046). Smoking, alcohol intake, herniorrhaphy, an epididymal cyst, medication and illicit drugs were not associated with any significant semen variables, testicular volume or circulating reproductive hormones. BMI had a significantly negative correlation with semen volume (r = -0.12, P = 0.048), sperm output (r = -0.13, P = 0.02), serum LH (r = -0.16, P = 0.002), inhibin B (r = -0.16, P < 0.001), testosterone (r = -0.23, P < 0.001) and DHT (r = -0.22, P < 0.001) and a positive correlation with 3αD (r = 0.13, P = 0.041) and DHEA (r = 0.11, P = 0.03). Second semen samples compared with the first semen samples in the 287 participants who provided two samples, with no significant bias by Bland-Altman analysis. Testis volume was significantly correlated positively with sperm concentration (r = 0.25, P < 0.001) and sperm output (r = 0.29, P < 0.001) and inhibin B (r = 0.42, P < 0.001), and negatively correlated with serum LH (r = -0.24, P < 0.001) and FSH (r = -0.32, P < 0.001). SCSA was inversely correlated with sperm motility (r = -0.20, P < 0.001) and morphology (r = -0.16, P = 0.005). WHO semen reference criteria were all met by only 52 men (14.4%). Some criteria were not met at first analysis in 15-20% of men, including semen volume (<1.5 ml, 14.8%), total sperm output (<39 million, 18.9%), sperm concentration (<15 million/ml, 17.5%), progressive motility (<32%, 14.4%) and morphologically normal sperm (<4%, 26.4%), while all five WHO criteria were not met in four participants (1.1%). LIMITATIONS AND REASONS FOR CAUTION: This was a large cohort study; however, potential for recruitment bias still exists. Men who did not participate in the testicular evaluation study (n = 282) did not differ from those who did (n = 423) with regard to age, weight, BMI, smoking or circulating reproductive hormones (LH, FSH, inhibin B, T, DHT, E2, E1, DHEA, 3α-diol, 3ß-diol), but were significantly shorter (178 versus 180 cm, P = 0.008) and had lower alcohol consumption (P = 0.019) than those who did participate. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrated the feasibility of establishing a birth cohort to provide a relatively unbiased insight into population-representative sperm output and function and of investigating its determinants from common exposures. While varicocele, cryptorchidism and obesity may impact on human testicular function, most common drug exposures and the presence of epididymal cysts appear to have little adverse impact, and this study suggests that discrepancies from the WHO reference ranges are expected, due to its derivation from non-population-representative fertile populations.

KATNB1 in the human testis and its genetic variants in fertile and oligoasthenoteratozoospermic infertile men.

Oligoasthenoteratozoospermia (OAT) is a phenotype frequently observed in infertile men, and is defined by low spermatozoa number, abnormal spermatozoa morphology and poor motility. We previously showed that a mutation in the Katnb1 gene in mice causes infertility because of OAT. The KATNB1 gene encodes an accessory subunit of the katanin microtubule-severing enzyme complex; this accessory subunit is thought to modulate microtubule-severing location and activity. We hypothesized that KATNB1 may play a role in human spermatogenesis and that genetic variants in KATNB1 could be associated with OAT in humans. Using immunostaining, we defined the localization of the KATNB1 protein in human testes. KATNB1 was present during spermatid development, and in particular localized to the microtubules of the manchette, a structure required for sperm head shaping. To assess a potential association between genetic variants in the KATNB1 gene and infertile men with OAT, we performed direct sequencing of genomic DNA samples from 100 OAT infertile and 100 proven fertile men. Thirty-seven KATNB1 variants were observed, five of which had not previously been described. Ten variants were present only in OAT men, however, statistical analysis did not reveal a significant association with fertility status. Our results suggest that variants in the KATNB1 gene are not commonly associated with OAT infertility in Australian men.

Genetic variants in the human glucocorticoid-induced leucine zipper (GILZ) gene in fertile and infertile men.

Sertoli cell only (SCO) syndrome is the predominant histology for men with non-obstructive azoospermia (NOA) and is usually of unexplained aetiology. Studies in mouse models indicated that the X-linked gene glucocorticoid-induced leucine zipper (GILZ) is essential for survival and differentiation of spermatogonia, and meiosis. GILZ deficiency results in a rapid and progressive loss of germ cells with SCO tubules and sterility in adults. The role of GILZ in human fertility has not been examined. Here we show that GILZ is localized to spermatogonia and spermatocytes in the human testis in a pattern analogous to that seen in mice. To assess the potential for an association between GILZ variants and human infertility, we sequenced the entire protein-coding regions of the GILZ gene in 65 SCO and 87 fertile Australian men. We identified six genetic variants, three of which had not been reported previously. Three variants, 107018665 G>A, 107018485 C>G and 106959283 C>T, were found at a low frequency only in SCO men. Although none of the identified variants changed the protein code, sequence analysis indicated that two variants, 107018665 G>A and 107018485 C>G, would completely abolish the exonic splicing enhancer (ESE)-binding motifs for the splicing factors SF2/ASF and SC35 respectively. This result prompted an assessment of whether these two variants were associated with male infertility in a separate population of men. We used a PCR-based SNP detection approach to screen an additional 52 NOA and 153 fertile Australian men, and 86 SCO and 54 fertile American men. None of these men carried either of these two variants. The cumulative allelic frequency of these variants is less than 1% in SCO men and no association with fertility status was observed. Our study suggests that GILZ variants are not common causes of SCO and NOA in Australian or American men.

Changing subcellular localization of nuclear transport factors during human spermatogenesis.

Spermatogenesis requires progressive changes in gene expression mediated by hormonal and local factors. Regulated macromolecular movement between nuclear and cytoplasmic compartments enables these essential responses to changing extracellular cues, and dynamic production of the nucleocytoplasmic transporters and importin proteins, throughout gametogenesis in rodents implicates them as key mediators of germline differentiation. We examined normal adult human testis expression profiles of six importins plus five additional proteins involved in nucleocytoplasmic transport. Although most were detected in the nucleus during germline differentiation, importin α4 was exclusively observed in Sertoli and germ cell cytoplasm. Many proteins were present in round spermatid nuclei (importins α1, α3, ß1, ß3; exportin-1, Nup62, Ran, RanBP1, RCC1), and remarkable intense nuclear and/or nuclear-associated signals were detected for importin α1, importin α3 and Nup62 in spermatocytes. This study identifies conserved aspects of nucleocytoplasmic transport during spermatogenesis and extends our knowledge of the dynamic presence of these proteins, which indicates that they contribute to germ cell-specific cargo trafficking and potentially to other functions during human spermatogenesis. We also demonstrate for the first time that importin α3 is nuclear in spermatocytes, when exportin-1 is cytoplasmic, suggesting that nuclear transport is altered during meiosis.

GGN1 in the testis and ovary and its variance within the Australian fertile and infertile male population.

Mouse gametogenetin (Ggn) is a testis-enriched gene that encodes multiple spliced transcripts giving rise to three predicted protein isoforms: GGN1, GGN2 and GGN3. Of these, GGN1 has been linked to germ cell development. Based on the spatial and temporal expression pattern of GGN1 during mouse spermatogenesis, it has been proposed as a candidate human infertility gene. Here, we report the localization of GGN1 in the human testis and ovary compared with the mouse orthologue. Within the testis, GGN1 was confined to pachytene spermatocytes and spermatids. During mid-prophase GGN1 redistributes from a solely cytoplasmic localization to both cytoplasmic and nuclear in late prophase spermatocytes and round spermatids, and is ultimately incorporated into the sperm tail. Within both mouse and human ovaries, GGN1 was localized within granulosa cells. Lower levels of expression were observed in mouse oocytes and the cumulus cells. Furthermore, to define the level of sequence variation in the fertile population and to assess the potential for an association with male infertility, we sequenced the coding region of human GGN in 100 idiopathic oligospermic infertile and 100 control men. Fifteen genetic variants were identified, of which 10 had not previously been reported. No significant associations with fertility status were observed, suggesting that variance in the GGN gene are not a common cause of oligospermic infertility in Australian men.

Variability in total testosterone levels in ageing men with symptoms of androgen deficiency.

The aim of this study was to determine the variability in total testosterone (TT) levels in healthy, non-obese ageing men with symptoms of androgen deficiency. Men aged ≥55 years were recruited from the community. Morning TT levels were measured on four occasions in a 12-month period. In all, 96 men aged 62.7 ± 6.8 years were studied. Geometric mean values (95% confidence interval) of TT levels for the cohort at each time point were 14.5 (13.4-15.7), 14.6 (13.5-15.8), 15.5 (14.4-16.8) and 15.0 (13.9-16.2) nmol/L. The maximum intra-individual difference in TT between the four samples was 4.1 nmol/L (interquartile range: 2.9-6.4). Using the average of two baseline TT values reduces the maximum difference to 2.3 nmol/L (1.0-3.8). Only 1 of 25 men with a documented TT <10 nmol/L at baseline had TT levels <10 nmol/L at all subsequent time points. A single TT level is a reliable predictor of repeat measures taken within a 12-month period for a cohort of healthy ageing men with symptoms of androgen deficiency. However, given that the diagnostic criteria for androgen deficiency are, in part, predicated upon serum TT, there is sufficient intra-subject variability to warrant repeat sampling to confirm an initial low TT level. Using an average of two baseline TT values reduces this variability.

Androgen receptor gene expression in the rat central nervous system: Evidence for two mRNA transcripts.

Androgen receptor (AR) gene expression in the central nervous system (CNS) and peripheral tissues of male rats was examined using cDNA probes to measure AR mRNA by RNA (Northern) blot analysis and by in situ hybridization. Using a probe from the 5' untranslated region of the rat cDNA (AR-1), a single mRNA species of approximately 11 kb was seen in Northern blots of poly(A)(+) RNA from reproductive tissues, kidney, liver, and muscle. Using a probe from the 5' end of the coding region (AR-2), in addition to the 11-kb band, a novel transcript was seen in whole brain at about 9.3 kb. In poly(A)(+) RNA from dissected brain regions, the 9.3-kb transcript was predominant in the cortex, cerebellum, and brain stem, while in the hippocampus, both transcripts were expressed to a similar degree. AR mRNA levels increased two- to threefold in the prostate on Days 1 and 3 following castration but no significant change was seen in either CNS transcript in whole brain or cortex. Specific in situ hybridization of an (35)S-labeled AR-2 riboprobe was observed in brain regions known to bind radiolabeled androgens. We conclude that two AR RNA species exist in the adult male rat which differ in their 5' untranslated region and that the relative proportion of the two species varies between brain regions. In contrast to observations in the prostate, AR gene expression in the cerebral cortex is not regulated in the short term by androgen withdrawal.

Polymorphisms in the human cysteine-rich secretory protein 2 (CRISP2) gene in Australian men.

BACKGROUND: Cysteine-rich secretory protein 2 (CRISP2) is localized to the human sperm acrosome and tail. It can regulate ryanodine receptors Ca(2+) gating and binds to mitogen-activated protein kinase kinase kinase 11 in the acrosome and gametogenetin 1 (GGN1) in the tail. METHODS AND RESULTS: In order to test the hypothesis that CRISP2 variations contribute to male infertility, we screened coding and flanking intronic regions in 92 infertile men with asthenozoo- and/or teratozoospermia and 176 control men using denaturing HPLC and sequencing. There were 21 polymorphisms identified, including 13 unreported variations. Three SNPs resulted in amino acid substitutions: L59V, M176I and C196R. All were only present in a heterozygous state and found in fertile men. However, the C196R polymorphism was of particular interest as it resulted in the loss of a strictly conserved cysteine involved in intramolecular disulphide bonding. Screening of an additional 637 infertile men identified 23 heterozygous C196R men to give an overall frequency of 3.6%, compared with 3.4% in control men. The functional significance of the C196R polymorphism was defined using a yeast two-hybrid assay. The C196R substitution resulted in the loss of CRISP2-GGN1 binding. CONCLUSIONS: Although none of the many polymorphisms identified herein showed a significant association with male infertility, functional studies suggested that the C196R polymorphism may compromise CRISP2 function.