Gender susceptibility to COVID-19: a review of the putative role of sex hormones and X chromosome.

BACKGROUND: The recent emergence of COVID-19 poses a global health emergency. One of the most frequently reported data is sex-related severity and mortality: according to the last available analysis on 239,709 patients in Italy, lethality is 17.7% in men and 10.8% in women, with 59% of total deaths being men. Interestingly, the infection rate is lower in males than in females, with 45.8% and 54.2% of positive cases, respectively, suggesting that gender-related factor may worsen disease evolution. A tentative hypothesis to explain these findings is the role of angiotensin-converting enzyme 2 (ACE2) and serine protease TMPRSS2 involved in viral infection. PURPOSE: In this review, we summarize the available evidence pointing to gender-related differences in ACE2 and TMPRSS2 expression, from both genetic and endocrine points of view. RESULTS: Altogether, available evidence points toward two not-mutually exclusive mechanisms in gender susceptibility to COVID-19 by sex hormonal regulation of ACE2 and TMPRSS2. On one hand, ACE2 expression could be increased in women, either by estrogens or constitutively by X chromosome inactivation escape or by reduced methylation, providing a larger reservoir of ACE2 to maintain the fundamental equilibrium of RAS regulatory axis. On the other, low levels of androgens in women may keep at low levels TMPRSS2 expression, representing a further protective factor for the development of COVID-19 infection, despite the increased expression of ACE2, which represents the Trojan horse for SARS-CoV-2 entry. CONCLUSIONS: Both mechanisms consistently point to the role of sex hormones and sex chromosomes in the differential severity and lethality of COVID-19 in men and women.

Perfluorooctanoic acid alters progesterone activity in human endometrial cells and induces reproductive alterations in young women.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of ß-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

Perfluoroalkyl substances and bone health in young men: a pilot study.

PURPOSE: Perfluoroalkyl substances (PFAS) are a class of endocrine-disrupting chemicals. Toxicological studies indicate that PFAS accumulate in bone tissue and could cause alterations in bone metabolism. The primary objective of this study was to examine the association between PFAS exposure and bone status in a cohort of young men resident in a well-defined area with high PFAS environmental pollution. METHODS: Bone status was assessed in 117 subjects aged 18-21 by quantitative ultrasound (QUS) at the heel. Subjects underwent an accurate medical visit. Socio-demographic characteristics, lifestyle, and medical histories were collected. We also verified the interaction between PFAS and hydroxyapatite by computational modelling. The organic anion-transporting peptide (OATP), the putative transporter of PFAS, was evaluated by qPCR in bone biopsies from femoral heads discarded during arthroplasty in three male subjects. RESULTS: Exposed subjects showed significantly lower stiffness index, which resulted in lower t-score and higher prevalence of subjects at medium-high risk of fracture (23.6%) compared with controls (9.7%). Data from computational modelling suggested that PFOA exhibits a high affinity for hydroxyapatite, since the estimated change in free energy is in the order of that exhibited by bisphosphonates. Finally, we observed consistent expression of OATP1A2 gene in primary human osteoblasts. CONCLUSIONS: This is the first study reporting increased osteoporosis risk in young men exposed to PFAS and provide preliminary information on molecular mechanisms that could explain this observation, in agreement with previous studies on animal models and humans. However, these results must be interpreted with caution given the cross-sectional study design and the small number of cases.

E2F1 copy number variations contribute to spermatogenic impairment and cryptorchidism by increasing susceptibility to heat stress.

BACKGROUND: Copy number variations (CNVs) play an important role in the onset of several diseases, and recently research focused on the relationship between these structural variants and diseases of the reproductive tract, including male infertility and cryptorchidism. OBJECTIVES: To evaluate the contribution of copy number variations of E2F1 gene to idiopathic male infertility and the factors influencing expression of this gene. MATERIALS AND METHODS: We performed a retrospective study on 540 subjects recruited from September 2014 to February 2015. TaqMan CNV assay was used to analyze E2F1 CNV. Real-time PCR was used to assess E2F1 and HSP70 expression level in heat stressed and transfected cells with three E2F1 copies. RESULTS: We found a significant difference in the frequency of altered E2F1 copies in patients (12/343, 3.5%) compared with controls (0/197) (p = 0.005). Six patients with E2F1 CNV had history of cryptorchidism, but the prevalence between men with idiopathic infertility (6/243, 2.5%) and infertile men with history of cryptorchidism (6/100, 6.0%) was not statistically different (p = 0.1). E2F1 expression increased under heat stress conditions, especially in cells carrying more copies of gene and this was associated with increased expression of HSP70. DISCUSSION: Our data suggest that an abnormal E2F1 expression caused by multiple copies of E2F1 gene predisposes to the onset of infertility and that the risk further increases if subjects with altered E2F1 copies have stressful conditions, such as heat stress or history of cryptorchidism. CONCLUSION: This study shows a link between E2F1 CNV and male infertility, suggesting that the increased risk of spermatogenic impairment associated with higher E2F1 copies might be due to higher susceptibility to stressful conditions.

Osteocalcin, a bone-derived hormone with important andrological implications.

Increasing evidence disclosed the existence of a novel multi-organ endocrine pathway, involving bone, pancreas and testis, of high penetrance in energy metabolism and male fertility. The main mediator of this axis is undercarboxylated osteocalcin (ucOC), a bone-derived protein-exerting systemic effects on tissues expressing the metabotropic receptor GPRC6A. The recognized effects of ucOC are the improvement of insulin secretion from the pancreas, the amelioration of systemic insulin sensitivity, in particular in skeletal muscle, and the stimulation of the global endocrine activity of the Leydig cell, including vitamin D 25-hydroxylation and testosterone production. The supporting evidence of this circuit in both animal and human models is here reviewed, with particular emphasis on the role of ucOC on testis function. The possible pharmacological modulation of this hormonal circuit for therapeutic aims is also discussed.

Sperm telomere length as a parameter of sperm quality in normozoospermic men.

STUDY QUESTION: Could sperm telomere length (STL) represent a novel parameter and biomarker of sperm quality? SUMMARY ANSWER: STL is associated with standard semen quality parameters and, more importantly, it is significantly associated with levels of DNA fragmentation and sperm protamination. WHAT IS KNOWN ALREADY: Telomeres are fundamental for genome integrity. Recent studies have demonstrated that STL increases with age and men with oligozoospermia have shorter sperm telomeres than normozoospermic men. STUDY DESIGN, SIZE, DURATION: Cohort study conducted from September 2014 to June 2015 on 100 subjects with normal standard semen parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: STL was measured indirectly by quantitative polymerase chain reaction using telomere/single-copy gene ratio, sperm DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay and protamination by aniline blue staining. Data were analyzed for determining the relationships between STL, standard semen parameters and DNA fragmentation and protamination. MAIN RESULTS AND THE ROLE OF CHANCE: Among standard semen parameters, STL was positively associated with progressive motility (P = 0.004) and vitality (P = 0.007). STL was significantly and negatively associated with sperm DNA fragmentation (P = 0.001) and significantly and positively associated with protamination (P = 0.002). The role of chance was limited and the findings have biological relevance and a pathophysiological explanation. LIMITATIONS, REASONS FOR CAUTION: For the present study, we deliberately selected only men with normozoospermia to better analyze whether STL might represent a biomarker of sperm quality beyond traditional sperm parameters. Additional studies in proven fertile men with normal sperm parameters are needed. WIDER IMPLICATIONS OF THE FINDINGS: The measurement of STL is a simple and rapid method that offers further information about the quality of sperm. The results of this study demonstrate that STL could be considered as an additional sperm parameter and opens new perspectives in the evaluation of the infertile male. Additional studies will clarify the significance of this parameter also as a prognostic biomarker in assisted reproduction. STUDY FUNDING/COMPETING INTERESTS: No external funding was either sought or obtained for this study. There are no conflicts of interest to be declared.

The Klinefelter syndrome is associated with high recurrence of copy number variations on the X chromosome with a potential role in the clinical phenotype.

The Klinefelter syndrome (KS) is the most frequent sex chromosomal disorder in males, characterized by at least one supernumerary X chromosome (most frequent karyotype 47,XXY). This syndrome presents with a broad range of phenotypes. The common characteristics include small testes and infertility, but KS subjects are at increased risk of hypogonadism, cognitive dysfunction, obesity, diabetes, metabolic syndrome, osteoporosis, and autoimmune disorders, which are present in variable proportion. Although part of the clinical variability might be linked to a different degree of testicular function observed in KS patients, genetic mechanisms of the supernumerary X chromosome might contribute. Gene-dosage effects and parental origin of the supernumerary X chromosome have been suggested to this regard. No study has been performed analyzing the genetic constitution of the X chromosome in terms of copy number variations (CNVs) and their possible involvement in phenotype of KS. To this aim, we performed a SNP arrays analysis on 94 KS and 85 controls. We found that KS subjects have more frequently than controls X-linked CNVs (39/94, [41.5%] with respect to 12/42, [28.6%] of females, and 8/43, [18.6%] of males, p < 0.01). The number of X-linked CNVs in KS patients was 4.58 ± 1.92 CNVs/subject, significantly higher with respect to that found in control females (1.50 ± 1.29 CNVs/subject) and males (1.14 ± 0.37 CNVs/subject). Importantly, 94.4% X-linked CNVs in KS subjects were duplications, higher with respect to control males (50.0%, p < 0.001) and females (83.3%, p = 0.1). Half of the X-linked CNVs fell within regions encompassing genes and most of them (90%) included genes escaping X-inactivation in the regions of X-Y homology, particularly in the pseudoautosomal region 1 (PAR1) and Xq21.31. This study described for the first time the genetic properties of the X chromosome in KS and suggests that X-linked CNVs (especially duplications) might contribute to the clinical phenotype.

D-Aspartic acid stimulates steroidogenesis through the delay of LH receptor internalization in a mammalian Leydig cell line.

PURPOSE: Recent experimental evidence on non-mammalian animal models showed that D-Aspartic acid (d-Asp) administration increases testosterone levels through upregulation of StAR in Leydig cells. In this study, we aimed to investigate in vitro the signaling pathway associated with d-Asp stimulation in MA-10 murine Leydig cells. METHODS: MA-10 cells were stimulated with different concentrations of d-Asp, in presence or absence of hCG. Then total testosterone (T) levels in the culture medium were evaluated by electrochemiluminescence immunoassay, and StAR and LHR protein expressions were quantified by the means of Western blotting. LHR cellular localization after hormonal stimulation was assessed by immunofluorescence. RESULTS: Stimulation with the sole d-Asp did not induce any relevant increase of T release from cultured cells. On the other hand, stimulation with hCG induced significant increase of T (P = 0.045). Concomitant stimulation with hCG and d-Asp, at the concentration of 0.1 and 1 nM, induced additional and significant increase of released T (P = 0.03 and P = 0.04, respectively). StAR protein levels increased after concomitant stimulation with hCG and d-Asp 0.1 nM, compared with stimulation with the sole hCG (P = 0.02), whereas no variation in LHR protein expression was observed. Finally, d-Asp attenuated displacement of LHR staining, from cell membrane to cytoplasm, subsequent to hCG stimulation. CONCLUSIONS: In this study, we confirmed a steroidogenic role for d-Asp, in concert with hCG, on murine Leydig cells, which is mediated by an increase in StAR protein levels. In addition, we showed that the possible mechanism subtending the effect of d-Asp could rely on the modulation of LHR exposure on the cell membrane.

In young men sperm telomere length is related to sperm number and parental age.

STUDY QUESTION: What are the relationships between telomere lengths in leukocytes and sperm, sperm count and parents' age at conception in a group of apparently healthy subjects of the same age? SUMMARY ANSWER: Sperm telomere length (STL) is related to sperm count, it is lower in oligozoospermic than in normozoospermic men and it is directly related to parents' age at conception. WHAT IS KNOWN ALREADY: Leukocyte telomere length (LTL) decreases with age but STL increases and offspring of older fathers tend to have longer leukocyte telomeres. Only one study analyzed STL in relation to male fertility, and reported shorter telomeres in infertile versus fertile men. No data have been reported on STL in relation to parents' age at conception. STUDY DESIGN, SIZE, DURATION: Prospective study conducted from January to December 2012 of 18-19-year-old high school students. PARTICIPANTS/MATERIALS, SETTING AND METHODS: The volunteers were 81 apparently healthy subjects, including 61 with normozoospermia and 20 with idiopathic oligozoospermia. Leukocyte and sperm telomere length were measured by real-time PCR. Data were analyzed for determining the relationships between LTL, STL, sperm count and parents' age at conception. MAIN RESULTS AND THE ROLE OF CHANCE: Sperm and leukocyte telomere length were strongly correlated, but STL was significantly longer. A significant positive correlation between STL and total sperm number was found. STL was significantly lower in oligozoospermic than in normozoospermic men. Finally, we found a significant positive relationship between maternal age and both leukocyte and sperm telomere length and a significant positive relation between paternal age and STL in the offspring. The relative contributions of mothers' and fathers' ages to their offspring's telomere length could not be determined because of the high correlation between paternal and maternal ages. LIMITATIONS AND REASONS FOR CAUTION: Although consistent with previous findings, this is the first study on telomere length in oligo- and normozoospermic men and included a relatively low number of subjects. Our study was also restricted to young (18-19 year old) men, so future studies should determine whether our findings can be generalized to men at ages typically encountered at fertility centers. Future studies should also try to determine the possible effect of abstinence time and frequency of ejaculation with STL. WIDER IMPLICATIONS OF THE FINDINGS: Our study sheds new light on the association between STL and sperm count and on the inheritance of telomere length (in leukocytes and sperm) in relation to the parents' age at conception. Additional studies are needed to confirm these observations, to clarify if the association between shorter STL and damaged spermatogenesis represents a pathophysiological link, and to determine the effect on offspring telomere length of assisted reproduction techniques performed on couples of advanced age or where the man is oligozoospermic. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Italian Ministry of University and Research (grant no. 2009AMPA9C to C.F.) and Padova University (grant 2010 to A.D.R.). The authors have no competing interests to declare.

De novo 911 Kb interstitial deletion on chromosome 1q43 in a boy with mental retardation and short stature.

Patients with distal deletions of chromosome 1q have a recognizable syndrome that includes microcephaly, hypoplasia or agenesis of the corpus callosum, and psychomotor retardation. Although these symptoms have been attributed to deletions of 1q42-1q44, the minimal chromosomal region involved has not yet defined. In this report, we describe a 7 years old male with mental retardation, cryptorchid testes, short stature and alopecia carrying only an interstitial de novo deletion of 911 Kb in the 1q43 region (239,597,095-240,508,817) encompassing three genes CHRM3, RPS7P5 and FMN2.

Evidence for sexual differentiation of glia in rat brain.

It is well established that gonadal steroids mediate sexual differentiation of the brain via direct effects on neurons during a restricted critical period. In addition, estrogen can influence glial morphology in the adult brain, and in vitro studies suggest estrogen induces glial differentiation. However, there is a lack of in vivo evidence for steroid effects on glia during the critical period. We report here a hormone-mediated sexual differentiation of arcuate glia as early as Postnatal Day 1. Using glial fibrillary acidic protein immunoreactivity (GFAP-ir), we compared the responsiveness of astroglia in the rat arcuate nucleus among five hormonally different groups. The results indicate increased GFAP-ir cell surface area 24 hr after hormonal manipulation in castrate males compared to intact males, intact females (ANOVA; P < 0.01), and females injected with testosterone propionate (50 microg; ANOVA; P < 0.05). However, astroglia in intact males extended their processes significantly greater distances from the cell body compared to all other treatment groups (ANOVA; P < 0.01). The GFAP-ir cells were categorized into four distinct classes ranging from a simple bipolar to a fully stellate morphology. The frequency distribution of classes varied between groups with more stellate cells found in intact males. Finally, these sex differences in arcuate glia persisted into adulthood. We hypothesize that during the critical period, testosterone, or its metabolite estrogen, induce sexual differentiation of glia. We further hypothesize that in females glial cells remain partially undifferentiated and this may be important to glial plasticity seen in adult female arcuate.

Antiandrogen microimplants into the rostral medial preoptic area decrease gamma-aminobutyric acidergic neuronal activity and increase luteinizing hormone secretion in the intact male rat.

gamma-Aminobutyric acid (GABA)ergic neurons terminating in the rostral hypothalamus are stimulated by testosterone. To investigate whether this action is mediated locally through androgen receptors in the rostral hypothalamus, bilateral microcannulas (28 gauge) containing the androgen receptor antagonist, hydroxyflutamide (HF), were stereotaxically implanted into the rostral medial preoptic area (rMPA) just dorsal to the major population of GnRH cell bodies. Two days later, blood samples were collected for assay of LH, and animals were killed for determination of GABAergic neuronal activity in tissue dissected from the site of the implanted cannulas. Animals were decapitated either without treatment or 60 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, ip). The rate of GABA accumulation in the tissue after aminooxyacetic acid treatment was used as a measure of GABA turnover. Levels of messenger RNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67), the rate-limiting enzyme responsible for GABA synthesis also were measured by a microlysate ribonuclease protection assay. LH levels were significantly increased (1.8-fold) in HF-treated animals compared with controls. In the MPA, beneath the implant cannulas, GABA turnover was significantly reduced in HF-treated rats. There was no effect of treatment in the frontal cortex, which was used as a control region. Surprisingly, levels of messenger RNA for both GAD65 and GAD67 were significantly increased in HF-treated rats. The results indicate that GABAergic neurons terminating in the rostral hypothalamus are tonically stimulated by testosterone acting by means of androgen receptors localized in this region. These findings support the working hypothesis that androgen-sensitive GABAergic neurons in the rMPA mediate the negative feedback action of testosterone on GnRH secretion in the male rat.

GABAergic neuronal activity and mRNA levels for both forms of glutamic acid decarboxylase (GAD65 and GAD67) are reduced in the diagonal band of Broca during the afternoon of proestrus.

There is considerable evidence that GABAergic neurons play an important role in the regulation of gonadotropin-releasing hormone (GnRH) secretion, and that these neurons may mediate the feedback actions of gonadal steroids on GnRH neurons. The aim of the present study was to investigate whether endogenous changes in ovarian steroid secretion during the estrous cycle influenced GABAergic neuronal activity in the preoptic region of the hypothalamus, and in other steroid-sensitive brain regions. Intact, adult female rats were sacrificed at various times during the days of metestrus or proestrus. GABAergic neuronal activity was estimated by measuring the rate of accumulation of GABA in microdissected brain regions after pharmacological inhibition of GABA degradation. Concentrations of mRNA for both forms of glutamic acid decarboxylase (GAD65 and GAD67) were quantified in microdissected brain regions by a microlysate ribonuclease protection assay. In the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis (DBB(ovlt)), GABAergic neuronal activity was significantly reduced during the afternoon of proestrus compared with the morning of either proestrus or metestrus. In the lateral septal nucleus, GABAergic neuronal activity was significantly increased in the afternoon of proestrus compared with the morning. There were no significant effects of time of day or day of estrous cycle in the medial preoptic nucleus, median eminence, ventromedial nucleus, suprachiasmatic nucleus, medial septal nucleus, hippocampus (CA1 region), or cingulate cortex. In the DBB(ovlt), mRNA levels for both GAD65 and GAD67 were significantly reduced in the afternoon of proestrus compared with the afternoon of metestrus. By contrast, there was no change in GAD65 and GAD67 mRNA levels in the cingulate cortex at any of the times examined. These results demonstrate that GABAergic neuronal activity, and mRNA levels for both GAD65 and GAD67, are reduced in the DBB(ovlt) during the afternoon of proestrus. These results support the hypothesis that decreased GABAergic neuronal activity in this region plays a major permissive role in the generation and maintenance of the estrogen-induced LH surge.