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Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

Background: Reinke crystals are structures pathognomonic for Leydig cells, which have the important function of testosterone production and are vital for male reproductive health. These crystalline inclusions are thought to be of protein origin; however, the molecular composition has not yet been resolved., Objectives: This review summarizes all available information regarding Reinke crystal's characteristics and aims to produce a comprehensive guide for research on this topic as well as to determine and discuss potential Reinke-protein candidates., Methods: Pubmed was thoroughly searched for all publications regarding Reinke crystals and 137 publications were identified. All publications were surveyed and all relevant information was included in the review., Results: Along with the cytoplasm, structures that resemble Reinke crystals were also observed in the nucleus, suggesting that their formation depends only on protein concentration. Variations in tissue processing protocols could impact Reinke crystal microscopic visualization, which is an important factor in diagnosing Leydig cell disorders such as Leydig cell tumors. Reinke crystals appear to be hallmarks of normally differentiated, adult, Leydig or Leydig-like cells in humans, while some abnormal and nonhuman Leydig cells contain Reinke-like paracrystalline inclusions or crystalloids., Conclusions: These characteristics point to some differentially expressed proteins, which could be involved in Reinke crystal formation. Differential Reinke crystal and paracrystalline inclusion presence could also be due to small changes in protein structure or the cell environment. Further research is needed to solve the ongoing mystery of the Reinke crystal, which would enhance our knowledge of Leydig cell contribution in the pathogenesis of various male reproductive disorders and improve their diagnosis and treatment., (© 2022 American Society of Andrology and European Academy of Andrology.)

Cartilage differentiates in rat limb buds cultivated in a chemically defined protein-free medium in the same manner as in the richer serum-supplemented medium. We aimed to investigate the remaining differentiation potential of pre-cultivated limb buds by subsequent transplantation in vivo. Rat front (FLBs) and hind-limb buds (HLBs) were isolated from Fischer rat dams at the 14th gestation day (GD 14) and cultivated at the air-liquid interface in Eagle's Minimum Essential Medium (MEM) alone; with 5 μM of 5-azacytidine (5azaC) or with rat serum (1:1). Overall growth was measured seven times during the culture by an ocular micrometre. After 14 days, explants were transplanted under the kidney capsule of adult males. Growth of limb buds was significantly lower in all limb buds cultivated in MEM than in those cultivated with serum. In MEM with 5azaC, growth of LBs was significantly lower only on day 3 of culture. Afterwards, it was higher throughout the culture period, although a statistically significant difference was assessed only for HLBs. In transplants, mixed structures developed with the differentiated transmembranous bone, cartilage with enchondral ossification, bone-marrow, sebaceous gland, and hair that have never been found in vitro. Nerves differentiated only in transplants precultivated in the serum-supplemented medium. We conclude that pre-cultivation of LBs in a chemically defined protein-free medium does not restrict osteogenesis and formation of epidermal appendages but is restrictive for neural tissue. These results are important for understanding limb development and regenerative medicine strategies., (© 2022 The Authors. Anatomia, Histologia, Embryologia published by Wiley-VCH GmbH.)

Although DNA methylation epigenetically regulates development, data on global DNA methylation during development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle's Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O. Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3. That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.

Male reproductive development starts early in the embryogenesis with somatic and germ cell differentiation in the testis. The LIN28 family of RNA-binding proteins promoting pluripotency has two members-LIN28A and LIN28B. Their function in the testis has been investigated but many questions about their exact role based on the expression patterns remain unclear. LIN28 expression is detected in the gonocytes and the migrating, mitotically active germ cells of the fetal testis. Postnatal expression of LIN28 A and B showed differential expression, with LIN28A expressed in the undifferentiated spermatogonia and LIN28B in the elongating spermatids and Leydig cells. LIN28 interferes with many signaling pathways, leading to cell proliferation, and it is involved in important testicular physiological processes, such as cell renewal, maturation, fertility, and aging. In addition, aberrant LIN28 expression is associated with testicular cancer and testicular disorders, such as hypogonadotropic hypogonadism and Klinefelter's syndrome. This comprehensive review encompasses current knowledge of the function of LIN28 paralogs in testis and other tissues and cells because many studies suggest LIN28AB as a promising target for developing novel therapeutic agents.

Background: The bryophytes are a plant group that is smaller than and not as well known as the vascular plants. They are less used and are almost completely neglected in ethnobotanical studies. Traditional nativity scenes depicting the birth of Christ are commonly decorated with both vascular plants and bryophytes. The aim of this study was to document the diversity of decorative bryophytes sold during the Advent season at farmers' markets in Croatia (Southeastern Europe, Balkan Peninsula)., Methods: Twenty-eight farmers' markets in the two largest Croatian cities (Zagreb in the continental part and Split in the Mediterranean part) were studied in the search for local vendors selling bryophytes during the pre-Christmas season. The bryophytes collected were identified and analysed with respect to families, growth type, life forms and threat status., Results: Among 275 collected specimens, 43 moss and four liverwort species were identified. The mean number of species per vendor was 3.5. The most frequent species were Hypnum cupressiforme, Homalothecium sericeum and Ctenidium molluscum. Mats, wefts and tufts were the most common life-forms, while pleurocarpous prevailed over acrocarpous mosses, as they are usually pinnately branched and form large carpets, suitable for decorations. The overall selection of bryophytes and the decorations made of them were more diverse and abundant in inland Croatia, where 49 vendors at 15 farmers' markets sold goods containing 43 species. In Mediterranean Croatia, at six farmers' markets only 29 vendors sold goods, which contained 18 species. A considerable number of species that are less attractive to harvesters were collected non-intentionally, entangled in carpets of other, more frequent species. Among them, Rhodobryum ontariense and Loeskeobryum brevirostre are rare and insufficiently recorded in Croatia so far., Conclusions: The present study provided a first perspective on the use of bryophytes in traditional nativity scenes in Croatia and Southeastern Europe, contributing to scarce ethnobotanical documentation of the decorative use of bryophytes in Christmas festivities in Europe and globally., (© 2022. The Author(s).)

Background: Non-obstructive azoospermia (NOA) is a form of male infertility caused by disorders of the testicular parenchyma and impaired spermatogenesis. This study aimed to investigate the nature of Leydig cell changes in patients with NOA, especially whether their actual proliferation occurred., Methods: 48 testicular biopsies from infertile patients with NOA and 24 testicular biopsies originating from azoospermic patients suffering from obstructive azoospermia (OA) were included in the study. Leydig cells and their possible proliferative activity were analysed by immunohistochemistry and morphometry (stereology)., Results: Unlike in the OA group, Leydig cells in NOA patients were sometimes organised into larger clusters and displayed an abundant cytoplasm/hypertrophy. Moreover, significant fibrosis of the interstitial compartment was demonstrated in some NOA samples, often accompanied by inflammatory cells. Stereological analysis showed no increase/proliferation of Leydig cells; on the contrary, these cells decreased in number in the NOA group., Conclusions: The decrease in the number of Leydig cells can be explained by previous inflammatory changes within the testicular interstitium and consequent interstitial fibrosis. The interstitial fibrosis might have a deteriorating effect on Leydig cells.

A study on the headspace volatile organic compounds (VOCs) profile of native populations of Sideritis romana L. and Sidertis montana L., Lamiaceae, from Croatia is reported herein, to elucidate the phytochemical composition of taxa from this plant genus, well-known for traditional use in countries of the Mediterranean and the Balkan region. Headspace solid-phase microextraction (HS-SPME), using divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) or polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber, coupled with gas chromatography-mass spectrometry (GC-MS) was applied to analyze the dried aerial parts of six native populations in total. Furthermore, principal component analysis (PCA) was conducted on the volatile constituents with an average relative percentage ≥1.0% in at least one of the samples. Clear separation between the two species was obtained using both fiber types. The VOCs profile for all investigated populations was characterized by sesquiterpene hydrocarbons, followed by monoterpene hydrocarbons, except for one population of S. romana , in which monoterpene hydrocarbons predominated. To our knowledge, this is the first report on the VOCs composition of natural populations of S. romana and S. montana from Croatia as well as the first reported HS-SPME/GC-MS analysis of S. romana and S. montana worldwide.

The upsurge in male infertility over the last two decades, possibly due to environmental exposure, has raised significant interest, particularly boosted by reports from fertility clinics, which showed that chronic diseases and hereditary or other medical conditions might only partially explain current incidence of male infertility. Both environmental and occupational settings may have a significant role in exposure to complex mixtures of endocrine disruptors (ED), which play a major role in fertility disorders. The aim of this review is to give an insight into the current knowledge on exposure settings which may be associated with male infertility. Our study relied on a systematic search of PubMed, Scopus, and Web of Science for articles published between January 2000 and September 2020. It showed that some well documented factors associated with male infertility include smoking, and physiological disturbances or chronic diseases such as obesity and diabetes, which in turn, may also reflect lifestyle choices and environmental exposures, especially to EDs such as phthalates, bisphenols, pesticides, and flame retardants. However, the number of studies on the aetiology of male infertility is still too low in comparison with the size of affected population. Occupational health follow-ups and medical surveillance do not collect any data on male infertility, even though ED chemicals are part of many technological processes., (© 2021 Tihana Marić, Aleksandra Fučić, Anna Aghayanian, published by Sciendo.)

Aim: Research frequency of puerperal disorders in nulliparous women who had cesarean section in University Hospital Mostar in year 2004 and given results compared with nulliparus women who had vaginal mode of delivery., Patients and Methods: Research included 119 nulliparous women who had C-section in year 2004 (Tested group) and 119 nulliparous women who had vaginal mode of delivery (control group) taking in count time of delivery (first next one in delivery room protocol) and age (+/- 2 years). We analyzed type of puerperal disorders and their frequency in these groups and made comparison among them., Results: Frequency of puerperal disorders in nulliparous women delivered by cesarean section is higher increased compared with nulliparous women who had vaginal mode of delivery (chi2=15,015; p<0,001). In tested group was found significantly higher frequency of anemia (chi2=8,895; p=0,003) and puerperal fever (chi2=8,333; p=0,004). Higher risk of puerperal disorders in nulliparous women who had cesarean section was confirmed by odds ratio which was 4,183., Conclusion: Higher incidence of puerperal disorders in nulliparous women who had cesarean section is confirmation of higher risks for women with operative mode of delivery which is concerning due to increased trend of cesarean sections in our country and in the world.