Probes for INS

Study question: Can our newly developed testicular organoid (TO) growth platform advance the robustness of murine TOs? Summary answer: The platform resulted in more consistent TO histology. Moreover, improved germ cell survival was observed after a two-week culture with numbers comparable to fresh samples. What is known already: Organ cultures have traditionally been used for invitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Organoids made from testicular cell suspensions offer these features. Although TO cultures can result in organoids with compartmentalized testicular architecture, histological heterogeneity between individual TOs limits reproducibility of the results, offering unreliable readouts. Moreover, germ cell loss is characteristic during the reorganization phase. Study design, size, duration: Here, we tested a new TO growth platform. Firstly, the focus was put on improving germ cell survival in TOs during tubulogenesis in the first two weeks of culture. For this, four different growth media (A-D), supplemented with other combinations or concentrations of growth factors, were compared. Next, five cell seeding densities (I-V) were tested for their ability to recreate the testicular architecture in TOs in the selected culture media. Participants/materials, setting, methods: Testicular cells from 5 days old C57BL/6J mice were grown in our TO platform with alpha-MEM-based medium, previously found to support TO generation in mice (medium A). Three additional conditions were tested in their ability to improve germ cell survival during tubulogenesis (B-D). Finally, the ideal cell density (I-V) was determined based on histological resemblance to native tissue: one tubule-like structure and surrounding interstitium. Cellular reorganization and germ cell maintenance were characterized by (immuno)histochemistry. Main results and the role of chance: During short-term cultures of 2 weeks, testicular cells self-assembled and compacted into organoids in our platform. Interestingly, media B and D resulted in the highest amount of germ cells (p < 0.05), comparable to the fresh control. Particularly TOs cultured in medium D also exhibited the largest surface area, indicative for better in-vitro growth. Finally, TOs that were cultured in condition D had the best histology when grown at cell density IV and V (p < 0.05). Limitations, reasons for caution: Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells typically observed in organ culture, but also in TOs. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings: . The opportunities TOs offer to manipulate cells (genetic modification, inclusion and exclusion) are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity and drug discovery studies.

Infertility is a common and rapidly growing health issue around the world. The genetic analysis based on the infertile population is crucial for intervention and treatment.To find candidate gene locus led to azoospermia in Chinese multi-ethnic groups and provide theoretical guidance for the diagnosis of genetic diseases to progressively aggravated infertility patients and sterile offspring with ART.The study based on whole-exome sequencing (WES) was presented for genetic characteristic analysis of multi-ethnics and identification of variants related to infertility in Xinjiang area of China.The frequency of pathogenic variants showed significant ethnic differences among four main ethnics in Xinjiang. The population structure analysis confirmed that the Hui was close to the Han population, the Kazak was close to the Uygur population, and there are three ancestry components in the four ethnics. In addition, ten candidate variants potentially regulated azoospermia were detected, and KNTC1 (rs7968222: G > T) was chosen to validate the association. Through the analysis in the valid group, the frequency of rs7968222 (G > T) has a significant difference in the azoospermia population (11.76%, 8/68) and normospermia population (4.63%, 35/756) (P < 0.001). Interestingly, the proportion of people with abnormal follicle-stimulating hormone (FSH) level in the group carrying rs7968222 (G > T) was significantly higher than non-carriers (P < 0.05). Therefore, rs7968222 may regulate spermatogenesis through affecting hormone level.Our study establishes the genetics analysis of Northwest China and finds a candidate gene locus KNTC1 (rs7968222: G > T), which is one of the genetic susceptibility factors for male azoospermia.

Folliculogenesis is a tightly coordinated process essential for generating a fertilization-competent gamete while also producing gonadal hormones that sustain endocrine function. In vitro follicle growth systems have been critical to our understanding of key events in folliculogenesis, such as gonadotropin-independent and -dependent growth, steroid hormone production, and oocyte growth and maturation (cytoplasmic and meiotic). Although there are several successful follicle culture strategies, the following protocol details an encapsulated in vitro follicle growth (eIVFG) system for use with mouse ovarian follicles. eIVFG is performed with alginate hydrogels, which are biologically inert, maintain cell-to-cell interactions between granulosa cells and the oocyte, and preserve follicle architecture as found in the ovary. The system supports follicle growth, development, and differentiation from the early primary follicle to the antral follicle stage. Moreover, post-folliculogenesis events including meiotic maturation, ovulation, and luteinization are also supported. Importantly, the culture of secondary follicles has successfully resulted in viable pups after blastocyst transfer. This alginate-based eIVFG system is versatile and has broad applications as a tool for interrogating the fundamental biology of the ovarian follicle in a controlled manner, a screening platform for toxicity and bioactivity, and a potential fertility preservation method for endangered species as well as humans.

Fetal growth restriction (FGR) significantly contributes to neonatal and perinatal morbidity and mortality. Currently, there are no effective treatment options for FGR during pregnancy. We have developed a nanoparticle gene therapy targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF1) to correct fetal growth trajectories. Using the maternal nutrient restriction guinea pig model of FGR, an ultrasound-guided, intraplacental injection of nonviral, polymer-based hIGF1 nanoparticle containing plasmid with the hIGF1 gene and placenta-specific Cyp19a1 promotor was administered at mid-pregnancy. Sustained hIGF1 expression was confirmed in the placenta 5 days after treatment. Whilst increased hIGF1 did not change fetal weight, circulating fetal glucose concentration were 33%-67% higher. This was associated with increased expression of glucose and amino acid transporters in the placenta. Additionally, hIGF1 nanoparticle treatment increased the fetal capillary volume density in the placenta, and reduced interhaemal distance between maternal and fetal circulation. Overall, our findings, that trophoblast-specific increased expression of hIGF1 results in changes to glucose transporter expression and increases fetal glucose concentrations within a short time period, highlights the translational potential this treatment could have in correcting impaired placental nutrient transport in human pregnancies complicated by FGR.

What are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing?We discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues.Previous studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues.We collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas.We collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas.Our data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas.The datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122.Our study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles.Our study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas.This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Study question To elucidate whether AZF screening is indicated in men with proven non-obstructive azoospermia (NOA) or severe oligozoospermia (&lt;5x106 sperm/mL) who concomitantly harbor chromosomal abnormalities. Summary answer Some chromosomal aberrations are concomitant with AZF microdeletions, and hence Y chromosome microdeletion (YCM) screening is necessary for these patients. What is known already In the era of assisted reproduction, finding cost-minimization strategies in infertility clinics without affecting the quality of diagnosis and treatment is becoming one of the top-priority topics for future research. Formulating definitive guidelines concerning the indications for paraclinical testing could be one of the strategies. Accordingly, definitive guidelines for AZF screening do not exist especially in men who also have chromosomal abnormalities. The current clinical practice is to decide per case whether to pursue further molecular testing, with the accompanying added psychological and socio-economic burden. Study design, size, duration An observational retrospective cohort study of 10,388 consecutive cases from a tertiary infertility referral center (Royan institute, Tehran, Iran) over the last 12 years. A comprehensive literature review was also performed to summarize scientific evidence. Participants/materials, setting, methods The study recruited the largest cohort of men with primary infertility (NOA or severe oligozoospermia) ever presented who underwent screening for cytogenetic abnormalities and YCMs using sequence-tagged sites-polymerase chain reaction (STS-PCR) with a primer set covering the AZF regions according to the EAA/EMQN guidelines, as part of the infertility workup prior to ART. Main results and the role of chance In total, one-third of men with YCMs concomitantly had an abnormal karyotype (excluding those with heteromorphisms) (169/565; 29.9%, 95% CI: 26.3-33.8). In respect to the frequency of YCMs, our findings suggest that the cases diagnosed with 46,X,add(X) with incidence of 1.8% (3/169; 95% CI: 0.6-5.1); 45,X and mosaic forms: 45,X/46,X,inv(Y)(p11.2q12); 45,X/46,X,del(Y); 45,X,der(Y;Autosome); 45,X/46,X,idic(Y)(p11.2); 45,X/46,XY,r(Y); and 45,X/46,X,idic(Y)(q11.2) (19/169; 11.2%, 95% CI: 7.3-16.9); and inv(Y)(p11.2q12) (2/169; 1.2%, 95% CI: 0.3-4.2) should also be referred for AZF deletion screening, as data suggests they are accompanied with YCMs. Limitations, reasons for caution The extension of the outcomes beyond the described population could introduce concerns on appropriate medical management. Confirmatory studies in other geographic/ethnic groups are still necessary to reach a consensus regarding the outcomes. Wider implications of the findings It has been recommended that all men with NOA who have chromosomal abnormalities, except those with 46,XY/45,X karyotype, do not need AZF testing. The results reflect a crucial need for reconsidering whether YCM testing is indicated in the population of men with certain karyotypic abnormalities. Trial registration number Not Applicable