Influence of substance use on male reproductive health and offspring outcomes.

The prevalence of substance use globally is rising and is highest among men of reproductive age. In Africa, and South and Central America, cannabis use disorder is most prevalent and in Eastern and South-Eastern Europe, Central America, Canada and the USA, opioid use disorder predominates. Substance use might be contributing to the ongoing global decline in male fertility, and emerging evidence has linked paternal substance use with short-term and long-term adverse effects on offspring development and outcomes. This trend is concerning given that substance use is increasing, including during the COVID-19 pandemic. Preclinical studies have shown that male preconception substance use can influence offspring brain development and neurobehaviour through epigenetic mechanisms. Additionally, human studies investigating paternal health behaviours during the prenatal period suggest that paternal tobacco, opioid, cannabis and alcohol use is associated with reduced offspring mental health, in particular hyperactivity and attention-deficit hyperactivity disorder. The potential effects of paternal substance use are areas in which to focus public health efforts and health-care provider counselling of couples or individuals interested in conceiving.

Cessation of chronic delta-9-tetrahydrocannabinol use partially reverses impacts on male fertility and the sperm epigenome in rhesus macaques.

OBJECTIVE: To determine whether discontinuation of delta-9-tetrahydrocannabinol (THC) use mitigates THC-associated changes in male reproductive health using a rhesus macaque model of daily THC edible consumption. DESIGN: Research animal study. SETTING: Research institute environment. PATIENT(S): Adult male rhesus macaques (age, 8-10 years; n = 6). INTERVENTION(S): Chronic daily THC edible administration at medically and recreationally relevant contemporary doses followed by cessation of THC use. MAIN OUTCOME MEASURE(S): Testicular volume, serum male hormones, semen parameters, sperm deoxyribonucleic acid (DNA) fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing of sperm DNA. RESULT(S): Chronic THC use resulted in significant testicular atrophy, increased gonadotropin levels, decreased serum sex steroid levels, changes in seminal fluid proteome, and increased DNA fragmentation with partial recovery after discontinuation of THC use. For every increase of 1 mg/7 kg/day in THC dosing, there was a significant decrease in the total testicular volume bilaterally by 12.6 cm3 (95% confidence interval [CI], 10.6-14.5), resulting in a 59% decrease in volume. With THC abstinence, the total testicular volume increased to 73% of its original volume. Similarly, with THC exposure, there were significant decreases in the mean total testosterone and estradiol levels and a significant increase in the follicle-stimulating hormone level. With increasing THC dose, there was a significant decrease in the liquid semen ejaculate volume and weight of coagulum; however, no other significant changes in the other semen parameters were noted. After discontinuing THC use, there was a significant increase in the total serum testosterone level by 1.3 ng/mL (95% CI, 0.1-2.4) and estradiol level by 2.9 pg/mL (95% CI, 0.4-5.4), and the follicle-stimulating hormone level significantly decreased by 0.06 ng/mL (95% CI, 0.01-0.11). Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. Whole genome bisulfite sequencing identified 23,558 CpGs differentially methylated in heavy-THC vs. pre-THC sperm, with partial restoration of methylation after discontinuation of THC use. Genes associated with altered differentially methylated regions were enriched for those involved in the development and function of the nervous system. CONCLUSION(S): This is the first study demonstrating that discontinuation of chronic THC use in rhesus macaques partially restores adverse impacts to male reproductive health, THC-associated sperm differentially methylated regions in genes important for development, and expression of proteins important for male fertility.

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol.

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.

The effects of delta-9-tetrahydrocannabinol exposure on female menstrual cyclicity and reproductive health in rhesus macaques.

OBJECTIVE: To determine the dose-dependent effect of contemporary marijuana exposure on female menstrual cyclicity and reproductive endocrine physiology in a nonhuman primate model. DESIGN: Research animal study. SETTING: Research institute environment. ANIMALS: Adult female rhesus macaques (6-12 years of age; n = 8). INTERVENTIONS: Daily delta-9-tetrahydrocannabinol (THC) edible at medically and recreationally relevant contemporary doses. MAIN OUTCOME MEASURES: Menstrual cycle length (MCL), anti-Müllerian hormone, prolactin, basal follicle-stimulating hormone (FSH), estradiol (E2) and progesterone, luteinizing hormone (LH), and thyroid-stimulating hormone. RESULTS: The average before THC weight was 6.9 kg (standard deviation, 0.8), and at the highest THC dosing, the average weight was 7.2 kg (standard deviation, 0.8). With increasing THC dosing, MCL and FSH concentrations increased, while basal E2 concentration was stable. The average MCL concentration increased 4.0 days for each mg/7 kg/day of THC (95% CI, 1.4-6.6 days). Follicle-stimulating hormone concentration increased significantly with increasing THC dose, 0.34 ng/mL for each mg/7 kg/day of THC (95% CI, 0.14-0.57 ng/mL). No significant trends were observed between THC dosing and average basal progesterone, anti-Müllerian hormone, prolactin, LH, or thyroid-stimulating hormone concentrations. CONCLUSIONS: In rhesus macaques, a dose response toward increased MCL and basal FSH concentrations but plateau of basal E2 and LH concentrations was observed with increasing THC dosing, suggesting ovulatory dysfunction. Further studies are needed to determine the effects of a longer duration of exposure and whether the significant increase in MCL and FSH concentrations results in reduced fecundity.

Postpubertal spermatogonial stem cell transplantation restores functional sperm production in rhesus monkeys irradiated before and after puberty.

BACKGROUND: Cancer treatment of prepubertal patients impacts future fertility due to the abolition of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but no studies have involved cytotoxic treatment before puberty and transplantation after puberty, which would be the most likely clinical scenario. OBJECTIVES: To evaluate donor-derived functional sperm production after SSC transplantation to adult monkeys that had received testicular irradiation during the prepubertal period. MATERIALS AND METHODS: We obtained prepubertal testis tissue by unilaterally castrating six prepubertal monkeys and 2 weeks later irradiated the remaining testes with 6.9 Gy. However, because spermatogenic recovery was observed, we irradiated them again 14 months later with 7 Gy. Three of the monkeys were treated with GnRH-antagonist (GnRH-ant) for 8 weeks. The cryopreserved testis cells from the castrated testes were then allogeneically transplanted into the intact testes of all monkeys. Tissues were harvested 10 months later for analyses. RESULTS: In three of the six monkeys, 61%, 38%, and 11% of the epididymal sperm DNA were of the donor genotype. The ability to recover donor-derived sperm production was not enhanced by the GnRH-ant pretreatment. However, the extent of filling seminiferous tubules during the transplantation procedure was correlated with the eventual production of donor spermatozoa. The donor epididymal spermatozoa from the recipient with 61% donor contribution were capable of fertilizing rhesus eggs and forming embryos. Although the transplantation was done into the rete testis, two GnRH-ant-treated monkeys, which did not produce donor-derived epididymal spermatozoa, displayed irregular tubular cords in the interstitium containing testicular spermatozoa derived from the transplanted donor cells. DISCUSSION AND CONCLUSION: The results further support that sperm production can be restored in non-human primates from tissues cryopreserved prior to prepubertal and post-pubertal gonadotoxic treatment by transplantation of these testicular cells after puberty into seminiferous tubules.

Restoration of functional sperm production in irradiated pubertal rhesus monkeys by spermatogonial stem cell transplantation.

BACKGROUND: In male pre-pubertal cancer patients, radiation and chemotherapy impact future fertility by eradication of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but only a small percentage of spermatozoa produced were of donor origin. Transient hormone suppression with a GnRH antagonist (GnRH-ant) enhanced spermatogenic recovery from transplanted SSCs. OBJECTIVES: To evaluate donor-derived and endogenous spermatogenic recovery after SSC transplantation into irradiated monkeys and to test whether hormone suppression around the time of transplantation facilitates spermatogenic recovery. MATERIALS AND METHODS: Testes of 15 adult rhesus monkeys were irradiated with 7 Gy and 4 months later transplanted, to one of the testes, with cryopreserved testicular cells containing SSCs from unrelated monkeys. Monkeys were either treated with GnRH-ant for 8 weeks before transplantation, GnRH-ant from 4 weeks before to 4 weeks after transplantation, or with no GnRH-ant. Tissues were harvested 10 months after transplantation. RESULTS: Two of the 15 monkeys, a control and a pre-transplantation GnRH-ant-treated, showed substantially higher levels of testicular spermatogenesis and epididymal sperm output in the transplanted side as compared to the untransplanted. Over 84% of epididymal spermatozoa on the transplanted side had the donor genotype and were capable of fertilizing eggs after intracytoplasmic sperm injection forming morulae of the donor paternal origin. Low levels of donor spermatozoa (~1%) were also identified in the epididymis of three additional monkeys. Transplantation also appeared to enhance endogenous spermatogenesis. DISCUSSION AND CONCLUSION: We confirmed that SSC transplantation can be used for restoration of fertility in male cancer survivors exposed to irradiation as a therapeutic agent. The success rate of this procedure, however, is low. The success of filling the tubules with the cell suspension, but not the GnRH-ant treatment, was related to the level of colonization by transplanted cells.

Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring.

Testicular tissue cryopreservation is an experimental method to preserve the fertility of prepubertal patients before they initiate gonadotoxic therapies for cancer or other conditions. Here we provide the proof of principle that cryopreserved prepubertal testicular tissues can be autologously grafted under the back skin or scrotal skin of castrated pubertal rhesus macaques and matured to produce functional sperm. During the 8- to 12-month observation period, grafts grew and produced testosterone. Complete spermatogenesis was confirmed in all grafts at the time of recovery. Graft-derived sperm were competent to fertilize rhesus oocytes, leading to preimplantation embryo development, pregnancy, and the birth of a healthy female baby. Pending the demonstration that similar results are obtained in noncastrated recipients, testicular tissue grafting may be applied in the clinic.

Can myocardial remodeling be a useful surrogate predictor of myocardial iron load? A 3D echocardiographic multicentric study.

The relationship between myocardial iron load and eccentric myocardial remodeling remains an under-investigated area; it was thought that remodeling is rather linked to fibrosis. This study aims to determine whether or not measures of remodeling can be used as predictors of myocardial iron. For this purpose, 60 patients with thalassemia were studied with 3D echocardiography and myocardial relaxometry (T2*) by Cardiac MRI. 3D derived sphericity index was significantly higher in patients with myocardial iron load. It was correlated with T2* with a 100% sensitivity and specificity (cut-off value of 0.34) to discriminate between patients with and without myocardial iron overload.

Effect of menstrual cycle phase and hormonal treatments on evaluation of tubal patency in baboons.

BACKGROUND: We evaluated whether menstrual cycle phase influences the assessment of tubal patency by hysterosalpingography (HSG) in baboons. METHODS: Retrospective analysis of baseline tubal patency studies and serum estradiol (E2 ) and progesterone (P4) values obtained from female baboons used as models for development of non-surgical permanent contraception in women. The main outcome measure was bilateral tubal patency (BTP) in relationship with estradiol level. RESULTS: Female baboons (n = 110) underwent a single (n = 81), two (n = 26), or three (n = 3) HSG examinations. In 33/142 (23%) HSG examinations, one or both tubes showed functional occlusion (FO). The median E2 in studies with BTP (49 pg/mL) was significantly higher than in those studies with FO (32 pg/mL, P = .005). Among 18 animals with repeat examinations where serum E2 changed from <60 to ≥ 60 pg/mL, 13 results changed from FO to BTP (P = .0001). No sets showed a change from BTP to FO with an increase in estradiol. CONCLUSION: In baboons, functional occlusion of the fallopian tube is associated with low estradiol levels, supporting a role for estrogen-mediated relaxation of the utero-tubal junction.

Characterization of tubal occlusion after transcervical polidocanol foam (PF) infusion in baboons.

OBJECTIVE: Our long-term goal is to develop a nonsurgical method of fallopian tubal occlusion for the purpose of permanent contraception. We have previously demonstrated that transcervical administration of 5% polidocanol foam (PF) can create tubal occlusion in macaques but that multiple treatments are required. In this study, we assessed the efficacy of various regimens of PF with and without depomedroxyprogesterone acetate (DMPA) (to control ovarian cycle phase) in the baboon. STUDY DESIGN: Adult cycling female baboons were evaluated for tubal patency by hysterosalpingography and then received a transcervical infusion of PF with (+) or without (-) an intramuscular injection of DMPA (3.5 mg/kg). Two concentrations of PF were compared: 1% [(+) DMPA, n=5; (-) DMPA, n=3] and 5% [(+) DMPA, n=4; (-) DMPA, n=3]. Controls received (+) DMPA (n=2) or (-) DMPA, (n=3) only. The reproductive tracts were removed 1-3 months after treatment for examination. RESULTS: No fallopian tubal occlusion was observed in negative controls (±DMPA). Histologic complete tubal occlusion was observed in 3/8 of females treated with 1% PF and in 6/7 treated with 5% PF. Histologic evaluation suggested that 1% PF is associated with prolonged chronic inflammation (more than 2-3 months), while 5% treatment eliminates the epithelial lining, at least focally, and resolves into complete occlusion within 1-2 months. This pattern of complete occlusion was seen in all 4 females that received 5% PF (+DMPA) and in 2/3 that received 5% PF (-DMPA). CONCLUSION: In a baboon model of transcervical permanent contraception, a single treatment with 5% PF resulted in complete tubal occlusion more reliably (85%) than 1% PF (38%). Cotreatment with DMPA may improve treatment results with 5% PF but requires additional study. IMPLICATIONS: A finding that a single transcervical treatment with 5% PF can occlude the fallopian tubes of baboon supports further study of this approach as a novel strategy for permanent contraception for women.

Blockade of tubal patency following transcervical administration of polidocanol foam: initial studies in rhesus macaques.

OBJECTIVE: To demonstrate the feasibility of polidocanol foam (PF) as a nonsurgical method of female permanent contraception using a nonhuman primate model. STUDY DESIGN: Four groups of adult female rhesus macaques underwent either transcervical treatment with 5% PF directly into the uterine cavity, treatment with inert (methylcellulose, MF) foam or no treatment followed by removal of the reproductive tract for histologic evaluation. Untreated animals were included in Group 1 (n=3). Group 2 animals (n=4) were treated once with MF. Group 3 (n=7) received a single, and Group 4 (n=5) received multiple monthly treatments with PF; in these 2 groups, baseline tubal patency was assessed either laparoscopically by chromopertubation (CP) or by hysterosalpingography. RESULTS: Group 1 (untreated) and Group 2 (MF) animals had normal tubal histology. In contrast, Group 3 and 4 females treated with PF showed evidence of tubal damage. In Group 4, bilateral tubal blockade was noted on CP after two (n=2) or three (n=3) treatments. Histologic analysis confirmed complete tubal occlusion (loss of epithelium, fibrosis) in three of these animals, and one showed significant tubal damage localized to the intramural segment. Nontarget (cervix, vagina, endometrium, ovary) reproductive tissues were unaffected. While similar tubal changes were observed after a single treatment (Group 3), endometrial hemorrhage was also noted as an acute change. CONCLUSION: PF is a promising candidate agent for nonsurgical permanent female contraception. The histologic features of PF occlusion are confined to the intramural portion of the tube. IMPLICATIONS: This study in rhesus macaques supports further development of transcervical administration of PF as a nonsurgical approach to permanent contraception. A nonsurgical method could reduce risks and costs associated with surgical female sterilization and increase access to permanent contraception.

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model.

The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.

Synchronisation of canine germinal vesicle stage oocytes prior to in vitro maturation alters the kinetics of nuclear progression during subsequent resumption of meiosis.

Inhibition of meiosis before in vitro maturation (IVM) can improve meiotic competence in immature mammalian oocytes. Therefore, meiosis-inhibiting agents were evaluated singularly for the ability to arrest and synchronise germinal vesicle (GV) stage canine oocytes, and the most effective treatments were combined to improve meiotic resumption rates. Oocytes cultured in 2 ng mL(-1) oestradiol (E2), 10 IU mL(-1) eCG, or both (EG) for 72 h resulted in significantly fewer oocytes resuming meiosis in EG than the control, E2, or with eCG. Oocytes cultured in 50 or 100 micromol L(-1) of butyrolactone 1 or roscovitine (ROS) for up to 48 h did not resume meiosis nor increase subsequent meiotic resumption rates following IVM. A combination of 50 micromol L(-1) ROS and EG treatment for 48 h significantly increased the proportion of canine oocytes in meiotic arrest. More importantly, following 48 h of IVM, ROS+EG-treated oocytes demonstrated a dramatic increase in the ability to resume meiosis compared with the non-treated controls (51.3 +/- 8.2% and 10.8 +/- 4.5%, respectively; P < 0.05). These data indicate that chemical and biological meiotic inhibitors are effective at inducing GV arrest in canine oocytes. Furthermore, these inhibitors are reversible and beneficial to subsequent meiotic resumption in vitro.