Cryobiology and fertility preservation: a perspective on past, current and future studies.

Cryopreservation has been used over many decades for the maintenance of viable biological specimens. Its expansion into the area of fertility preservation has been a natural outcome of the increased risks to human fertility from diseases, such as cancer and its treatment protocols, including radiation and chemo-therapy, and the general lifestyle trend to later marriages. The use of assisted reproductive techniques (ART) in preserving fertility have benefitted significantly from new scientific approaches, such as cryostorage, in which live cells and tissues are stored at low temperatures and revived when necessary. This review focuses on "cryopreservation science monitoring in reproductive biomedicine" to evaluate knowledge, trends, driving forces, impetus, and emerging technologies in order to draw a future roadmap for this field. Our analysis of the field of cryobiology emphasizes the significance of strategic planning of cryobiology research to support more its extensive use in therapeutics in the future. The Royan Institute (Tehran, Iran) recognises this need and has developed a strategic plan to engage in multidisciplinary research on the application of cryobiology, including cryobioengineering, in disease mitigation. We hoped that this study can help improve the quality and quantity of public discourse and expert awareness of the role for cryopreservation in fertility preservation within ART. DOI: 10.54680/fr23410110112.

Evaluation of Apoptotic Markers and Tissue Histology Indicate a Slight Advantage of Slow Freezing Method over Vitrification for Sheep Ovarian Tissues.

BACKGROUND: An efficient cryopreservation method is very important for preserving the fertility of sheep ovarian tissues. OBJECTIVE: To compare the slow freezing method and vitrification for cryopreservation of sheep ovarian tissues. METHODS: Dissected cortex fragments from ten sheep ovaries were used for the comparative study. Cryopreserved and control tissues were cultured for 24h and then evaluated according to follicular morphology and apoptotic assessment. RESULTS: In both slow freezing and vitrification methods, normal follicles were reduced when compared to the non-frozen control group. There were significantly more abnormal follicles with vitrification than with slow freezing method (P < 0.05). However, there were no significant differences with regard to apoptotic gene expression or the percentage of Caspase3 positive follicles among cryopreserved and control groups. CONCLUSION: A slight advantage of the slow freezing method was observed over vitrification for cryopreservation of sheep ovarian tissue fragments.

Tissue Engineered Human Amniotic Membrane Application in Mouse Ovarian Follicular Culture.

Since folliculogenesis requires a powerful cell-matrix interaction, natural scaffolds seem to be needed for follicular culture. Human amniotic membrane (HAM) offers promise as a support of in vitro ovarian follicular culture. HAM was decellularized with trypsin and EDTA. DNA and histology assays were performed to determine the elimination rate of genomic components. Cyto-biocompatibility of decellular AM (DAM) was verified by the cell viability (MTT) test. The small parts of intact amniotic membrane (IAM) and DAM were coated on the bottom of 96-well and each well was filled with 150 µL of base medium. Mouse primary-secondary (PS) follicles were separated to three groups: 1-culture in base medium (Control), 2-culture on IAM and 3-culture on DAM. Follicular size, morphology, viability, estradiol production and genes expression were evaluated and IAM group showed better growth and development in follicle culture. The viability rate and estradiol production in both experimental groups were statistically higher than the Control. Gdf9, Bmp15 and Cx37 were found to have higher expression levels in IAM group. Also, maximum apoptotic and survival indexes were determined in Control and IAM groups, respectively. Finally, IAM provides a better protective environment for mouse PS follicular culture that can reduce apoptosis level.

High level of intracellular sperm oxidative stress negatively influences embryo pronuclear formation after intracytoplasmic sperm injection treatment.

This study evaluates the relationship between sperm intracellular reactive oxygen species (ROS; H2 O2 , O2 ), DNA fragmentation (DF), low mitochondria membrane potential (MMP) of sperm and normal pronuclear formation among intracytoplasmic sperm injection (ICSI) patients. Semen samples were obtained from 62 infertile male who were candidates for ICSI treatment. After sperm processing, metaphase II (MII) oocytes were injected, and the mean percentages of intracellular ROS, MMP and DF were evaluated using flow cytometry. The mean percentages of pronuclear formation and zygote score (Z) were also recorded, and Pearson, Spearman's rank correlation coefficient and Kruskal-Wallis tests were applied to analyse the data. The amounts of sperm intracellular H2 O2 and O2-˙ had significant positive correlation with low MMP (P < 0.01). The intracellular ROS had a negative correlation with pronuclear formation (P < 0.05), and its effect was higher than 66.66%. In addition, the mean percentages of neither H2 O2 nor O2-˙ affected the quality of pronuclear embryos (Z-score). This study shows that although high levels of both sperm intracellular H2 O2 and O2-˙ in ICSI patients have deleterious effect on sperm MMP, only H2 O2 may interfere in pronuclear formation.

Relationship of seminal reactive nitrogen and oxygen species and total antioxidant capacity with sperm DNA fragmentation in infertile couples with normal and abnormal sperm parameters.

The objective of this study was to investigate the association between the amount of superoxide anion, peroxynitrite as oxidative stress (OS) markers and total antioxidant capacity (TAC) with sperm DNA fragmentation in infertile men with abnormal semen parameters. Semen samples were obtained from 102 infertile couples and divided into groups with normal and abnormal semen parameters according to the World Health Organization (WHO). Peroxynitrite and superoxide anions were detected using spectrofluorometric assays combined with 2,7 dicholorofluorescein (DCF)-DA and 4-chloro-7-nitrobenzo-2-oxa -1, 3-diazole (NBD-CL). Colorimetric assay was used for evaluation of TAC, while DNA fragmentation was studied by using sperm chromatin dispersion test. Superoxide anion, peroxynitrite and DNA fragmentation were significantly higher in infertile couples with abnormal semen parameters as compared to infertile couples with normal semen (P < 0.01). TAC was significantly lower in infertile men with abnormal semen parameters (P < 0.01). There was also a significant positive correlation between OS markers with sperm DNA fragmentation (r = 0.59, P < 0.01 and r = 0.67, P < 0.01, respectively). We have found that imbalance between superoxide anion and peroxynitrite with antioxidant capacity in infertile men with abnormal sperm parameters is associated with higher sperm DNA fragmentation.

Effects of different cryoprotectant combinations on primordial follicle survivability and apoptosis incidence after vitrification of whole rat ovary.

This study was aimed at obtaining best vitrification conditions for preservation of primordial follicles after vitrification of whole ovarian tissue of rats. Ovaries of prepubertal ~5-week old female Wistar rats were divided randomly into 7 groups: Control (non-vitrified), V1 (EG+DMSO), V2 (EG+PROH), V3 (DMSO+PROH), V4 (EG+DMSO+Sucrose), V5 (EG+PROH+Sucrose) and V6 (DMSO+PROH+Sucrose). Control and vitrified-warmed samples were sectioned serially and stained either with HE or anti and pro active caspase-3 kit. The number of intact follicles in different stages of development was lower and the number of atretic and apoptotic follicles was higher in vitrification groups than those of the control group. Cryoprotectant combinations in V4 group showed better follicular preservation especially for primordial follicle. V3, V4 and V5 were best cryoprotectant mixtures, after the control group, according to the number of atretic follicles but the incidence of apoptotic primordial follicles was lowest in V3, V4 and V6. Incidence of apoptosis and the number of atretic follicles were lowest in V3 and V4 groups, and there was better primordial follicle preservation and survivability in VIV group. Thus, the combination of EG + DMSO with sucrose appears to be better suited for vitrification of whole ovarian tissue of rats.

Expression of maturation genes and their receptors during in vitro maturation of sheep COCs in the presence and absence of somatic cells of cumulus origin.

The purpose of this study was to investigate the effects of somatic cells of cumulus origin (sCC) on gene expression and maturation of cumulus oocyte complexes (COCs) in vitro. Good quality (i.e., healthy-looking) isolated sheep COCs were randomly divided into two treatment groups: control (COC with no sCC) and coculture (COC with sCC). Nuclear maturation statuses of oocytes were assessed after 27 hours of in vitro culture. Moreover, the expression levels of growth differentiation factor 9 (GDF9), bone morphogenetic protein (BMP)15, BMP6, bone morphogenetic protein receptor II (BMPRII), activin like kinase 5 (ALK5) (transforming growth factor ß receptor 1: TGFßR1), ALK6 (BMPR1b), activin A receptor, type IIB (ActRIIB), and ALK3 (BMPR1a), as well as hyaluronan synthase 2 (HAS2) and prostaglandin endoperoxide synthase 2 (Ptgs2) in the COCs were assessed in both treatment groups after 3 h and 27 h of culture. The results showed that the proportion of metaphase II (MII) stage oocytes was significantly higher in the coculture group compared with the controls (77.21%±1.17 vs. 67.49%±1.80; P<0.05). The relative expressions of BMPRII, ALK6, and ActRIIB in control group and GDF9 and ActRIIB in coculture group showed significant differences during culture as assessed by real time polymerase chain reaction (P<0.05). The mean expression levels of BMPRII, ALK5, ALK6, and ActRIIB mRNA were decreased in the coculture group compared with those in the control group after 27 h of culture (P<0.05). In conclusion, we propose that in vitro maturation of sheep COCs alone disrupted the normal gene expression levels of both TGFß ligands and receptors, and also reduced the maturation rate. Coculture with sCC enhanced the maturation rate of oocytes concomitantly with reduced gene expression levels of a number of TGFß ligands and receptors.

Synchronizing cell cycle of goat fibroblasts by serum starvation causes apoptosis.

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5-year-old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.

Effect of mono-(2-ethylhexyl) phthalate (MEHP) on resumption of meiosis, in vitro maturation and embryo development of immature mouse oocytes.

The purpose of this study was to evaluate the effect of MEHP on in vitro maturation of mouse oocytes and resulting embryo development. Denuded oocytes (DO) were cultured in maturation medium supplemented with 0, 50, 100, 200 and 400 microM levels of MEHP for 24 h. The matured oocytes then were fertilized and cultured for 4 days. The percentage of Germinal Vesicle (GV) stage oocytes were significantly higher in 200 and 400 microM MEHP treatment comparing to the control (P < 0.05). The proportion of oocytes that progressed to the metaphase II (MII) stage was significantly decreased by adding of MEHP in a dose related pattern. The 2-cell embryo formation was significantly decreased with 400 microM treatments than the control. Moreover with further culture in experimental groups none of the embryos comparing to that of the control group were developed to morulla stage (P < 0.05). These results indicate that MEHP could negatively modulate mouse oocyte meiotic maturation in vitro and embryo development, suggesting possible risks for human and other mammalians reproductive health.

Computerized three-dimensional reconstruction of cartilage canals in chick tibial chondroepiphysis.

Cartilage canals have been extensively investigated, in particular as to their mode of formation, morphologic distribution, function and fate. We studied the morphological pattern of the cartilage canals of the right upper chondroepiphysis of the tibia of chick embryos, Hamburger-Hamilton stages 35-42, in serial sagittal wax sections and in reconstructions made with AutoCAD software. The spatial arrangement of the canals is presented in a series of drawings made according to computerized images. The canals were penetrated from three distinct surfaces: the anterior and superior surfaces of the tubercle and the posterior surfaces of the medial and lateral condyles. Immediately after entering, nearly all the canals were extended toward the medial or lateral aspects of the chondroepiphysis, from which no canals took their origin. All of the cartilage canals connected with the perichondrium, and their branching did not follow a specific pattern. The condylar canals did not unite with the tubercular ones.

Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells.

Embryonic stem (ES) cells can differentiate spontaneously into various lineages in vitro. However, spontaneous commitment of ES cells to the adipocyte lineage is rare. In the present study, bone morphogenic protein-4 (BMP-4) is described as a factor inducing adipocyte differentiation from ES cells at a high rate. For this reason, ES-cell-derived embryoid bodies (EBs) in suspension cultures were exposed to different doses of BMP-4 for 5 days before they were plated onto gelatin-coated tissue culture plates. Moreover, the effect of serum-containing and serum-free media in three different combinations was assessed. Plated EBs, stained with Sudan Black and processed for transmission and scanning electron microscopy, were observed daily for adipocyte formation. Treatment with BMP-4 resulted in the appearance of adipocyte clusters in EBs' outgrowth, depending on the doses applied. Early in differentiation, many small fat droplets were observed in adipocytes, while later on they coalesced and formed a few large fat droplets. Adipocyte clusters had a fibrillar and vascular stroma, and each adipocyte was surrounded with a reticular external lamina. Furthermore, the appearance and development of adipocytes and their changes following 2-3 weeks of starvation mimicked live adipose tissue. In fact, understanding the biological activity of growth and differentiation factors is needed to regulate and direct stem cell differentiation to specific cell types in vitro.

In vitro maturation of fresh and frozen-thawed mouse round spermatids.

Both initiation and maintenance of spermatogenesis are hormonally regulated by follicle stimulating hormone (rFSH) and testosterone. Co-culture systems also have important roles in the maintenance of spermatogenic cells. In this study, the effects of FSH and testosterone, co-culture system with Vero cells and co-culture supplemented with the hormones for maturation of frozen-thawed spermatids were determined. Testicular cells were suspended from the testis of National Medical Research Institute (NMRI) male mice and divided into two parts. The first aliquot of suspension was allocated for using as fresh and the rest was quickly cryopreserved. The frozen specimens were thawed and washed using Dulbecco modified Eagle's minimum essential medium (DMEM) medium. The fresh specimens were cultured in four groups: control (cultured on DMEM with 10% FBS), hormone (cultured on a medium supplemented with rFSH and testosterone), co-culture (cultured on Vero cells) and co-culture + hormone (cultured on Vero cells combined with rFSH and testosterone). The frozen-thawed specimens were cultured accordingly. The number of spermatids was recorded daily and the survival rates of each group were evaluated using Trypan blue test. The results showed that the number of the elongating spermatids was increased during the first day of the culture of fresh hormone, co-culture and co-culture + hormone groups. Viability rates of all kinds of the spermatid reduced during the 96 h of culturing. Our findings showed that the addition of hormone could support cell viability better than the co-culture. They also confirmed that the fresh round spermatid cells can progress into elongating and elongated spermatid only within the first 2 days of the culture in hormone, co-culture and co-culture + hormone groups. In the frozen-thawed specimens no extra significant increase in the number of cells was observed.

Sciatic nerve transection in neonatal rats induces apoptotic neuronal death in L5 dorsal root ganglion.

Transection of a peripheral nerve in neonatal rats induces death of the axotomized neurons which may be due to either necrosis or apoptosis. In the present investigation, neuronal cell death in L5 dorsal root ganglion was evaluated after unilateral sciatic nerve transection in rats at 1, 3, 5, 7 and 10 days age. After 5 days, right (experimental) and left (control) dorsal root ganglia in all groups were removed, fixed, processed and embedded for either light or electron microscopy. Normal nucleoli were counted in paraffin embedded serial sections, and correction factors for split and multiple nucleoli were applied as well as the physical disector. The number of neurons in the right dorsal root ganglia, as compared with the controls, was significantly lower in all groups, and the percentage of the reduction at 1, 3, 5, 7 and 10 days was 32.4, 27.2, 23.8, 22.8 and 21.8% respectively. On the other hand, the results of neuronal counts using the disector method showed 34.0, 25.7, 20.2, 20.0 and 14.2% reduction in the number of neurons at 1, 3, 5, 7 and 10 days, respectively. The microscopic and ultrastructural results indicated that there were typical morphological changes similar to those of apoptosis, including condensed basophilic nuclei, formation of nuclear caps, cell shrinkage and apoptotic body formation. We concluded that there is an increase in apoptosis in dorsal root ganglia following sciatic nerve axotomy with the greatest neuronal loss on postnatal day 1.

Chromomycin A3 staining as a useful tool for evaluation of male fertility.

PURPOSE: Our purpose was to investigate the association between percentage chromomycin A3 (CMA3) positivity of spermatozoa with some sperm parameters and in vitro fertilization rate. METHODS: Spermatozoa were collected from 139 men, washed in PBS, fixed in methanol/glacial acetic acid (3:1), and then spread on slides. CMA3 positivity is expressed as the percentage in 200 spermatozoa. RESULTS: Percentage of CMA3 positivity showed not only a negative correlation with fertilization rate but also a significant difference between fertilizing and nonfertilizing patients. Moreover, percentage of CMA3-positive spermatozoa showed a negative correlation with count and percentage motility and a positive correlation with percentage of abnormal morphology. Percentage of CMA3 positivity also had a positive correlation with some abnormalities of head such as amorphous and macrocephaly. Ultrastructural study showed chromatin unpackaging in high CMA3-positive semen samples in comparison with low CMA3-positive semen samples. CONCLUSION: There is a close relationship among fertilization rate, sperm parameters, and CMA3 positivity and CMA3 could be considered as a useful tool for evaluation of male fertility prior to infertility treatment.

Effect of Vero cell coculture on the development of frozen-thawed two-cell mouse embryos.

PURPOSE: Our purpose was to evaluate the beneficial effects of long-term coculture of Vero cells on the development of frozen-thawed two-cell mouse embryos. METHODS: Two-cell mouse embryos were frozen slowly with 1,2-propandiol and sucrose as cryoprotectants and thawed rapidly, followed by stepwise dilution. Vero cells were cultured in drops of RPMI 1640 to establish monolayers. Frozen-thawed embryos were cultured alone (control) or cocultured with Vero cells. The rate of development in both groups was compared. RESULTS: After 4 days of culture, significantly more embryos in coculture were developed to expanded blastocysts (61 vs 37% for controls; P < or = 0.0001). In addition, on the fifth day of cultivation, more embryos in coculture showed the potential of hatching from the zona pellucida (26 vs 7% in controls; P < or = 0.0001). The rate of degeneration in coculture was also much lower than in controls (6 and 15%, respectively). CONCLUSIONS: Coculture of cryopreserved preimplantation-stage embryos with Vero cells seems to be a useful tool to eliminate the postthaw deleterious effect of freezing and also to obtain better-quality embryos appropriate for transfer.