Association between female reproductive health and mancozeb: systematic review of experimental models

Abstract: Mancozeb is a widely used fungicide approved for use in agriculture in many countries with long persistence in the environment and consequent bioaccumulation in tissues and biological fluids. Despite the large amount of studies published in recent years, the relationship between mancozeb exposure and female reproductive health is not fully elucidated. In order to summarize current evidence on mancozeb exposure and female reproductive disease, we performed a systematic review of literature. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to make this review. An adapted version of the National Toxicology Program’s Office of Health and Assessment and Translation (OHAT) framework was used to evaluate the risk of bias. Electronic search on two databases (PubMed and Scopus) was used to find experimental studies (in vitro and in vivo) on mancozeb exposure. The database search identified 250 scientific articles, 20 of which met our inclusion criteria. Selected data were then reviewed and summarized in tables. Overall, mancozeb represents a hazard for female reproductive health, with different mechanisms of action. Undoubtedly more experimental and epidemiological studies are required to definitively validate mancozeb as reproductive toxicant

Generation of viable blastocysts from discarded human cleavage embryos

AbstractBackgroundWhile a relationship between embryo morphology, developmental ability, and genetic integrity exists, the selection of embryos with higher implantation potential remains a major challenge in assisted reproductive technology (ART). This study investigated blastocyst developmental competence and euploidy status in human embryos that had been classed as too poor quality to transfer (ET) or cryopreserve at the cleavage stage.Embryos were divided into three groups. Group 1 (n= 41) included good quality embryos from candidates of preimplantation genetic testing for aneuploidy (PGT-A). Groups II and III were the "rejected" supernumerary embryos, defined as suboptimal for ET or vitrification after morphological examination, with embryos randomly divided between the groups. Group II embryos (n= 31) were cultured up to the day 3 cleavage stage, when they were biopsied and fixed. Group III embryos (n= 27) were cultured up to the day 5 blastocyst stage, when they were evaluated for morphology and chromosomal status. Chromosomal status in all groups was assessed by multi-color fluorescence in situ hybridization (FISH) for chromosomes 13, 18, 21, X, and Y.ResultsEuploidy rates in groups I, II, and III were 56.1%, 38.7%, and 55.5 %, respectively. Among the blastocysts that developed from "rejected" embryos, 59.3% were classed as good quality. The most frequent chromosomal aneuploidy was related to the sex chromosome (22.2%). The mosaicism rate was not significantly different between the group II and III embryos (25.8% vs. 37.0%,p= 0.28).ConclusionIn conclusion, surplus poor-quality embryos rejected from clinical utilization at the cleavage stage may develop into viable blastocysts with normal chromosomal status for at least 5 chromosomes. Recovery of euploidy during poor-quality embryo transition from cleavage stage to blastocyst could provide an alternative choice for ET

Effects of simulated microgravity In vitro on human metaphase II oocytes: an electron microscopy-based study

The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria–vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocyte

Student learning performance in human anatomy using a virtual dissection table

In medical training it is fundamental to have a 3D understanding of human anatomy [1]. Body dissection is considered mandatory in most of the bio-medical schools. Medical schools around the world, constantly face the problem of availability of the cadavers. Apart from the classic methods (lectures, podcasts, atlas or photographs, models) technology advances made available new instruments to learn/teach 3D anatomy, which allow cadaverless dissection with the help of simulator software or virtual cadavers. The Anatomage® and Sectra® tables are advanced anatomy visualization systems, adopted by many of the world’s leading medical schools and institutions [2,3]. Here we report our experience with Anatomage® during the Academic Year 2016-17, in the Postgraduate Courses of Medical and Surgical Specialization, Master degrees in Medicine, Dental Medicine, Biology of Health and Nutrition, as well as Basic Degrees in Nursing and Biotechnology, of the University of L’Aquila. We enrolled 30 MD postgraduate students, and 440 undergraduate students. Both postgraduate and undergraduate medical students were allowed to handle the table. The other students assisted to class table demonstrations. An evaluation test was administered to all students at the end of the Courses. Our preliminary observations suggest that the use of virtual dissection table into the anatomical curriculum improves the learning student performance. Each student have a different set of needs, and the base line skills may be different. So, the teacher should take in consideration the variable scope of practice of the specific health professions. We are currently evaluating the efficacy of this technology at the end of the examinations. In the present preliminary report, we account with our results that are indicative of a positive impact on both basic and advanced anatomical learning

Contribution of light and electron microscopy in the identification of morphological alterations in large Japanese field mouse (Apodemus speciosus) testes exposed to low-dose-rate radiations

Ionizing radiation affects biological systems, resulting in an increased risk of cancer and mutagenesis. Male reproductive function is sensitive to ionizing radiation, with implications connected to infertility. Following the Nuclear Power Plants accident of Fukushima in 2011, there was great attention regarding exposure damage to low-dose-rate (LDR) radiation on the reproductive system. This preliminary study aimed to evaluate the role of light (LM) and transmission electron microscopies (TEM) to identify the potential effects of LDR radio-exposure on the morphology of large Japanese field mouse (Apodemus speciosus) testes living in the Fukushima Daiichi Nuclear Power Plant (FDNPP) ex-evacuation area. After collection samples were subjected to the standard preparative for LM and TEM. The testicular parenchyma was characterized by numerous seminiferous tubules, delimited by a thick and continuous basal lamina. Basally, the germinal epithelium presented round and pale spermatogonia, primary spermatocytes; while, more adluminally, round and elongated spermatids were at different phases of development. Pale and irregular Sertoli cells were interspersed among germ cells. Occasionally, cytoplasmatic holes interrupted the nuclear membrane integrity in spermatocytes and spermatids. Residual bodies were seen at the luminal surface. In conclusion, this study suggests that LM and TEM analysis are useful in evaluating potential morphological features in the male reproductive system after LDR exposure

Ultrastructural features of human metaphase II oocytes subjected to slow freezing or vitrification in an IVF program: a comparative analysis

During the past two decades important advances have been made in the field of assisted reproduction by using oocyte cryopreservation. However, mature (metaphase II) oocytes are very susceptible to cryodamage. In order to contribute to the identification of a cryopreservation protocol with minimal side effects on the oocyte structure and function, we evaluated and compared the subcellular features of human oocytes cryopreserved either with slow (controlled rate) freezing or vitrification (ultrarapid freezing). Supernumerary human metaphase II oocytes were donated by consenting patients enrolled in an IVF program. The age of these women ranged from 27 to 32 years old. The eggs were cryopreserved using slow freezing with 1.5M propanediol + 0.2M sucrose concentration or a closed vitrification system (Cryotip Irvine Scientific CA). Fresh oocytes were used as controls. Samples were fixed and prepared for light and transmission electron microscopy (LM and TEM) observations. By LM, all the oocytes were generally rounded, 90-100 microns in diameter, with regular ooplasm showing uniform distribution of organelles. By TEM, mitochondria-smooth endoplasmic reticulum (M-SER) aggregates were the most common structures found in all the oocytes fixed or cryopreserved within 3-4 hours after the retrieval. M-SER aggregates appeared instead partially replaced by mitochondria-vesicle complexes when oocytes were maintained in culture for a prolonged period of time. A slight to moderate vacuolization was found in the cytoplasm of the oocytes subjected to slow freezing. Slight microvacuolization was also found in vitrified oocytes, whereas vacuoles were almost completely absent in fresh controls. Amount and density of cortical granules (CGs) appeared abnormally reduced in all cryopreserved oocytes, irrespective of the protocol applied. In conclusion, it has been evidenced that prolonged stay in culture induces an intracellular membrane “recycling” in the oocytes, that causes the transformation of slender, anastomosed SER tubules into rounded vesicles surrounded by mitochondria, whose role is still uncertain. In addition, even though all cryopreservation protocols studied ensured a good overall preservation of the oocyte, vacuolization appears as a recurrent form of cell damage. This happens both during slow freezing and, at a lesser extent, during vitrification using a closed device. In addition, premature CG exocytosis was observed in both groups

Ultrastructural evaluation of mouse oocytes exposed in vitro to different concentrations of the fungicide Mancozeb

Mancozeb is a widely used fungicide, considered to be an endocrine disruptor. In vivo and in vitro studies evidenced its reproductive toxicity on mouse oocytes by altering spindle morphology, impairing oocyte maturation, fertilization, and embryo implantation. Mancozeb also induces dose-dependent toxicity on the ultrastructure of mouse granulosa cells, including chromatin condensation, membrane blebbing, and vacuolization. We evaluated the effects on the ultrastructure of mouse oocytes isolated from cumulus-oocyte complexes (COCs), exposed in vitro to increasing concentrations of mancozeb. COCs were matured in vitro with or without (control) low fungicide concentrations (0.001–1 μg/mL). All mature oocytes were collected and prepared for light and transmission electron microscopy. Results showed a preserved ultrastructure at the lowest doses (0.001–0.01 μg/mL), with evident clusters of round-to-ovoid mitochondria, visible electron-dense round cortical granules, and thin microvilli. Mancozeb concentration of 1 μg/mL affected organelle density concerning controls, with a reduction of mitochondria, appearing moderately vacuolated, cortical granules, and microvilli, short and less abundant. In summary, ultrastructural data revealed changes mainly at the highest concentration of mancozeb on mouse oocytes. This could be responsible for the previously described impaired capability in oocyte maturation, fertilization, and embryo implantation, demonstrating its impact on the reproductive health and fertility. © 2023 by the authors

Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes.

In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle

Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Effects of the pesticide Lindane on granulosa cell ultrastructure

The excessive exposure to pesticides in the Aral Sea area was correlated to the increased reproductive pathologies in those regions [1]. One of the principal chemical employed was the gamma-hexachlorocycloexane herbicide Lindane (L), a persistent organochlorine that may induces alterations in granulosa cell (GCs) survival [2, 3]. However, a comprehensive experimental study on the L-induced dose-effect morpho- logical alterations, has not yet addressed. Therefore, we studied by means of trans- mission and scanning electron microscopy, the morphological changes of mouse GCs, matured in vitro with increasing concentrations of L [4-6].GCs showed several dose-dependent changes, in respect to controls. In particular, we observed significant reduction of GC microvilli and decrease of cytoplasmic pro- cesses between adjacent GCs. In addition, peripheral aggregation of chromatin under the nuclear membrane, extensive plasma membrane blebbing, abundant GC remnants and cellular debris were also present. Mitochondria, endoplasmic reticula and Golgi apparatuses did not show significant changes. In conclusion, our results showed a dose-dependent toxicity of L on GCs, associated to morphological signs of apoptosis. Alterations of GCs may be associated to impaired oocyte competence and sterility [7].