A novel method for transmission electron microscopy study of cytoplasmic fragments from preimplantation human embryos

Transmission electron microscopy (TEM) is the main tool for exploring the intracellular damage and organelle distribution. The cause of producing embryo cytoplsamic fragmentation is not completely understood. Since the fragments have detrimental effects on embryo development, the ultrastructural analysis of fragments may play an important role in fragmentation etiology and in embryo development as well. There are no studies regarding the ultrastructure of fragments in transferable embryos, because the preparation for TEM is not vital and embryos are discarded inevitably. This study aims to introduce a new method for ultrastructural evaluation of fragments without damaging the human cleaving embryos

Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF. © 2016 American Society of Andrology and European Academy of Andrology

Ultrastructure of cytoplasmic fragments in human cleavage stage embryos

Purpose: The goal of this study was to evaluate the ultrastructure of cytoplasmic fragments along with the effect of cytoplasmic fragment and perivitelline space coarse granulation removal (cosmetic microsurgery) from embryos before embryo transfer on ART outcomes. Methods: One hundred and fifty intracytoplasmic sperm injection cycles with male factor infertility were included in this prospective study. Patients were divided into three groups of case (n = 50), sham (n = 50), and control (n = 50). Embryos with 10–50 % fragmentation were included in this study. Cosmetic microsurgery and zona assisted hatching were only performed in case and sham groups respectively. Extracted fragments were evaluated ultrastructurally by transmission electron microscopy (TEM). Rates of clinical pregnancy, live birth, miscarriage, multiple pregnancies, and congenital anomaly in the three groups were also compared. Results: Micrographs from TEM showed that mitochondria were the most abundant structures found in the fragments along with mitochondria-vesicle complexes, Golgi apparatus, primary lysosomes, and vacuoles. There were no significant differences in demographic characteristics, laboratory and clinical data, or embryo morphological features between the groups. The rate of clinical pregnancy in control, sham, and case groups had no significant differences (24, 18, and 18 %, respectively). The rates of live birth, miscarriage, multiple pregnancy, and congenital anomaly were also similar between the different groups. Conclusions: Our data demonstrated that cosmetic microsurgery on preimplantation embryos had no beneficial effect on ART outcomes in unselected groups of patients. As mitochondria are the most abundant organelles found in cytoplasmic fragments, fragment removal should be performed with more caution in embryos with moderate fragmentation

Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis

BACKGROUND: Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. METHODS: We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation. RESULTS: Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90-100 mum in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3-4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8-9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied. CONCLUSIONS: Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane "recycling" that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardenin

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

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes

Purpose: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. Methods: Samples were studied by light and transmission electron microscopy. Results: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. Conclusions: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism

Ultrastructural and morphometric evaluation of aged cumulus-oocyte-complexes

Maternal age is one of the most significant factors influencing oocyte quality (1). 35 years of age seems to be a watershed in reproductive potential. The aim of this study was to reveal the amount and distribution of specific ultrastructural organelles in human mature cumulus-oocyte-complexes belonging to women of different ages (<35 years old; ≥35 years old/ reproductive aging) and to evaluate their different response during 24 hours prolonged culture (defined as in vitro aging) (1). The samples were studied by light and transmission electron microscopy; a morphometric analysis of TEM data was performed (2). In all aged samples, the amount of mitochondria- smooth endoplasmic reticulum aggregates, cortical granules and microvilli decreased (p<0,05), while the amount of mitochondria-vesicle complexes increased up (p<0,05). Occasional vacuoles were found in oocytes from older women after in vitro aging. A significant (p<0,05) increase of zona pellucida thickness was linked to the donor age but not to in vitro aging. A re-compaction of cumulus cells was seen in in vitro aged samples. Morphometric data strongly confirmed our preliminary results (3) revealing that: i) reproductive aging and in vitro aging share specific ultrastructural features ii) In vitro aging can be consider a model for reproductive aging iii) young oocytes seem to be less sensitive to in vitro aging than older ones. The above results may represent a reliable background for further multidisciplinary studies regarding aged oocytes and may be also useful in clinical settings

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

Morphology and viability of human spermatozoa vitrified with a new, cryoprotectant-free, artificial seminal fluid

Cryopreservation is a process finalized to store tissues and cells at a very low temperature. The most common freezing protocols used for gamete preservation in Assisted Reproductive Technologies are slow freezing and vitrification (1). Vitrification combines ultrarapid cooling with high concentrations of cryoprotectants; it avoids, better than slow freezing, the formation of ice crystals. It has been demonstrated, however, that cryoprotectant addition may significantly reduce cell viability (2). This study was aimed to design a new, cryoprotectant-free, medium similar to normal human seminal fluid (SF) formulation (artificial seminal fluid; ASF), and to compare the cryoprotective potential of this medium with SF and Human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with swim-up technique and sperm suspensions were divided in four groups: fresh (controls); vitrified in HTF (Vit HTF); vitrified in patients’ SF (Vit SF); and vitrified in ASF (Vit ASF). To identify the effects of the different media we assessed sperm parameters of motility, viability and morphology after warming. Spermatozoa ultrastructure was also evaluated by scanning and transmission electron microscopy (SEM and TEM). The results showed that sperm motility, viability and normal morphology were significantly higher in Vit ASF than in Vit HTF. The same parameters were better in Vit ASF than in Vit SF, but only viability differed significantly. Deep cytoplasmic invaginations and folded tails were commonly observed by SEM in all vitrified sperms, but this alterations were more evident in Vit HTF and Vit SF than in Vit ASF. By TEM, acrosome damage, plasma membrane loss, chromatin vacuolation, disruption of mitochondria and adherence of several tail sections together were observed in all vitrified groups; the latter phenomenon, however, was more evident in Vit HTF and Vit SF than in Vit ASF. In conclusion, vitrification of human spermatozoa with ASF seems more effective in preserving sperm quality than Vit SF and, particularly, Vit HTF