Oocyte quality evaluation: a review of engineering approaches toward clinical challenges.

Although assisted reproductive technology has been very successful for the treatment of infertility, its steps are still dependent on direct human opinion. An important step of assisted reproductive treatments in lab for women is choosing an oocyte that has a better quality. This step would predict which oocyte has developmental competence leading to healthy baby. Observation of the oocyte morphological quality indicators under microscope by an embryologist is the most common evaluation method of oocyte quality. Such subjective method which relies on embryologist's experience may vary and leads to misdiagnosis. An alternative solution to eliminate human misjudging in traditional methods and overcome the limitations of them is always using engineering-based procedure. In this review article, we deeply study and categorize engineering-based methods applied for the evaluation of oocyte quality. Then, the challenges in laboratories and clinics settings move forward with translational medicine perspective in mind for all those methods which had been studied were discussed. Finally, a standardized process was presented, which may help improving and focusing the research in this field. Moreover, effective suggestion techniques were introduced that are expected they would be complementary methods to accelerate future researches. The aim of this review was to create a new prospect with the engineering approaches to evaluate oocyte quality and we hope this would help infertile couples to get a baby.

Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

CONTEXT: Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. AIMS: The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. METHODS: One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. KEY RESULTS: In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). CONCLUSIONS: Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. IMPLICATIONS: This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

Two leading international congresses in Iran in the era of COVID-19: 21st royan international twin congress, 4th international and 16th Iranian genetics congress.

In this report, we look at the challenges posed by the outbreak of COVID-19 and how the Executive Board of these two congresses succeeded in overcoming those challenges and holding two congresses. The approach for a large festival with different virtual setting components provided a suitable solution that led to exemplary achievements and created an appropriate model for future virtual or combined virtual and face-to-face events. These events proved that pandemic problems could not limit the organizers, pushing them to make better use of the facilities and turning this threat into an opportunity.

Fe3O4 magnetic nanoparticles improve the vitrification of mouse immature oocytes and modulate the pluripotent genes expression in derived pronuclear-stage embryos.

The vitrification of Germinal Vesicle (immature) oocytes is beneficial for preservation of fertility in cases involving reproductive problems. The use of nanoparticles (NP(s)) as vitrification aid is a novel approach towards improving vitrification efficiency. The efficacy of use of iron oxide (Fe3O4) nanoparticles as vitrification aid is reported in this paper. Immature oocytes from NMRI mice were collected and divided into non-vitrified (nVit), Vitrified (Vit) and Vitrified + NP (Vit+NP) groups. In the Vit+NP group, solutions containing Fe3O4 nanoparticles at three different concentrations (0.004%, 0.008% and 0.016% w/v) were separately added to the vitrification solution and their effects on the vitrification of the oocytes were compared. The concentration that was found to be best performing (0.004% w/v) was used in vitrification studies in subsequent experiments. Mitochondrial function, apoptosis incidence, ultrastructure alteration, nuclear maturity, embryo formation and genes expression (Nanog, Oct4, Cdx2, and Sox2) were evaluated in response to the addition of the nanoparticle solution during vitrification. Nuclear maturity of oocyte and embryo formation increased significantly (P ≤ 0.05) in the vitrified + NP group. Expression of Sox2 also increased significantly in both vitrified and vitrified + NP groups. While there was a significant increase in Oct4 expression in the vitrified group as compared to control, there was no significant difference between vitrified and Vit+NP groups. The expression of Cdx2 decreased significantly (P ≤ 0.05) in the Vit+NP group. From these observations, Fe3O4 nanoparticles could protect immature oocytes from cryodamages, positively affect vitrification and modulate the pluripotency of derived pronuclear-stage embryos.

Supplementation of granulosa cells conditioned medium with pyruvate and testosterone could improve early follicular development in cultured 1-day-old mouse ovaries.

In vitro activation of primordial follicles could serve as a safe method to preserve fertility in patients with cancer subjected to ovarian tissue cryopreservation during oncotherapy, however the culture medium for this purpose requires to be optimized. Granulosa cell conditioned medium (GCCM) has been recognized to enhance primordial follicle activation and the present study was conducted to understand whether addition of pyruvate, a combination of insulin, transferrin and selenium (ITS) or testosterone to GCCM could improve its efficiency in this regard. To this end, 1-day-old mouse ovaries were cultured in four different media including CON (control; containing GGCM only), PYR (containing GCCM plus pyruvate), ITS (containing GCCM plus ITS) or TES (containing GCCM plus testosterone) for 11 days. Furthermore, follicular dynamics and gene expression of factors involved in follicular development were assessed using histological examination and RT-PCR, respectively, on days 5 and 11 of culture. Pyruvate decreased follicular activation, but it enhanced the progression of follicles to the primary stage. Moreover, it upregulated Bmp15 and Cx37 (P < 0.05). In the ITS group, activation of follicles was not affected and total number of follicles was reduced by day 11 of culture. Additionally, ITS downregulated Pi3k, Gdf9, Bmp15 and Cx37 (P < 0.05). Although testosterone did not affect primordial follicle activation, it enhanced the development of follicles up to the preantral stage (P < 0.05). Furthermore, testosterone inhibited the expression of Pten but stimulated the expression of Gdf9 and Cx37 (P < 0.05). In conclusion, the present study revealed that inclusion of pyruvate and testosterone into GCCM could enhance the early development of follicles in cultured 1-day-old mouse ovaries.

Restoration of estrous cycles by co-transplantation of mouse ovarian tissue with MSCs.

This study investigates the effect of bone marrow (BM-MSCs) and visceral peritoneum (VP-MSCs)-derived mesenchymal stem cells on the transplanted ovary. VP-MSCs and BM-MSCs were obtained from green fluorescent protein-expressing mice (GFP+). Six- to eight-week-old female NMRI mice were divided into four experimental groups, autograft ovarian tissue fragments (AO), autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel (AO-H), autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel containing BM-MSCs (AO-HB) and autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel containing VP-MSCs (AO-HP). Intact ovary (IO) was the control group. The estrous cycles resumption time was monitored and at the third estrous cycle, the blood samples and grafted ovaries were evaluated using hormonal, histological and gene expression analysis. Onset of estrous cycles, especially at the second cycle, was earlier in AO-HB and AO-HP groups than in the AO-H group (P < 0.05). Moreover, E2 and FSH levels in AO-HB and AO-HP groups were returned to those of the intact group. However, folliculogenesis was still retarded as compared with the IO group. The gene expression of theca (Lhcgr, Cyp17a1, Gli2, Gli3 and Ptch1), granulosa (Amh and Fshr), oocyte (Zp3 and Gdf9), germ cells (Stella and Prdm1), angiogenesis (VEGF and bFGF) and apoptosis (Bax/Bcl2 and Caspase3) markers was similar in both AO-HB and AO-HP groups. Expression of Amh, Fshr, Gdf9 and VEGF increased only in the AO-HP group whereas expression of Ptch1 increased only in the AO-HB group, as compared with the AO group (P < 0.05). In conclusion, BM-MSCs or VP-MSCs can improve ovarian autotransplantation in mice with no superiority over each other.

Incorporation of arginine, glutamine or leucine in culture medium accelerates in vitro activation of primordial follicles in 1-day-old mouse ovary.

In vitro activation of primordial follicles provides cancer patients subjected to oncotherapy with a safe therapeutic strategy for fertility preservation, however a successful protocol for activation of primordial follicles in prepubertal patients has not yet been defined comprehensively. There is evidence that amino acids such as leucine, arginine and glutamine could stimulate the mammalian target of rapamycin (mTOR) pathway, which plays a pivotal role in primordial follicle activation. Nevertheless, there has been no report that elucidates the effect of these amino acids on in vitro development of ovarian follicles. Therefore, the present study was conducted to evaluate the effects of these amino acids and their combination on the formation and activation of primordial follicles in 1-day-old murine ovaries during an 11-day culture period. The experimental groups consisted of base medium (BM), base medium + arginine (ARG), base medium + glutamine (GLU), base medium + leucine (LEU) and base medium + a combination of arginine, glutamine and leucine (AGL). The proportions of different stages of ovarian follicles and gene expression of regulatory factors were assessed using histology and quantitative real-time PCR on days 5 and 11 of culture. The proportion of transitional and primary follicles was greater in all amino acid-treated groups compared with the BM group (P < 0.05). Moreover, leucine resulted in elevated expression of Gdf9 and Bmp15, and glutamine augmented the expression of Pi3k on day 11 of culture. In conclusion, the present study showed that inclusion of leucine, glutamine, arginine or their combination in the culture medium for murine ovarian tissue could accelerate the activation of primordial follicles and alter the expression of the corresponding factors.

Formation and activation induction of primordial follicles using granulosa and cumulus cells conditioned media.

Fertility preservation of prepubertal girls subjected to invasive cancer therapy necessitates defining protocols for activation of isolated primordial follicles. Granulosa (GCs) and cumulus cells (CCs) play pivotal role in oocyte development. Although GCs and CCs share some similarities, they differ in growth factors production. The current study was conducted to evaluate the effects of GCs, CCs and their conditioned media on mice primordial follicles activation. One-day-old mice ovaries were subjected to 6-day culture with base medium (BM), GC conditioned medium (GCCM), GC coculture (GCCC), CC conditioned medium (CCCM) or CC coculture (CCCC). Follicular growth and primordial to primary follicle transition was observed during 6-day culture, and follicular activation rate tended to be greater in GCCM than other groups (0.05

Proteomic analysis and microtubule dynamicity of human sperm in electromagnetic cryopreservation.

The proteomic changes, microtubule dynamicity, and quality parameters of human sperm were investigated during cryopreservation in an extremely low electromagnetic field (ELEF) condition. Semen samples were obtained from 210 healthy individuals with normospermia and then were divided into three experimental groups: fresh control, frozen control, and frozen ELEF group. Shotgun proteomics was performed to assess the identification of microtubule proteins of the sperm in experimental groups. Microtubule dynamicity, secondary, and tertiary structure modifications of tubulins, characteristics of transmission electron microscopy of sperm as well as sperm quality parameters were evaluated. The expression ratios of α- and ß-tubulins were significantly increased after cryopreservation compared with fresh control while this ratio was not significantly different in frozen ELEF group. The expression ratio of tubulin polymerization-promoting protein was significantly decreased after cryopreservation compared with fresh control. The length, width, and the activity of microtubule, secondary, and tertiary structures of tubulins, motility, and the viability of the sperm were decreased in frozen control as compared with fresh control. The microtubule activity, secondary, and tertiary structures of sperm tubulin in frozen ELEF group were higher than frozen control. Transmission electron microscopy of microtubules showed that the size of the width and length of the microtubules in frozen ELEF group were greater than frozen control. Motility, viability, and reactive oxygen species levels were improved in frozen ELEF group when compared with frozen control. While the microtubule dynamicity of the sperm was affected by the cryopreservation, this trait was improved during the electromagnetic cryopreservation resulted in better motility and viability.

Maturational gene upregulation and mitochondrial activity enhancement in mouse in vitro matured oocytes and using granulosa cell conditioned medium.

SummaryThe high miscarriage rates that result following transfer of embryos derived from in vitro maturation (IVM) of oocytes necessitate improvements in the processes involved. This study aimed to improve the quality of in vitro matured oocytes using granulosa cell conditioned medium (GCCM) as the culture medium. In this work, germinal vesicle (GV)-stage oocytes from NMRI mice were collected and cultured using three types of culture medium: Base medium (BM) (control), 50% granulosa cell conditioned medium (GCCM50) and 100% GCCM (GCCM100). After IVM, the mitochondria activity potential and viability of metaphase II (MII) oocytes were evaluated by JC-1 and trypan blue staining, respectively. Maturational gene expression levels of CyclinB1, Cdk1 and Gdf9 in the control, GCCM50 and GCCM100 samples were analyzed using real-time polymerase chain reaction (PCR). The viability rate of in vitro matured oocytes was highest in the GCCM50 group. JC-1 staining showed that GCCM50 enhances mitochondrial activity more than the other groups (P < 0.05). Gene expression levels of Cdk1 and Gdf9 were higher in the group with GCCM50 treatment, than in the control and GCCM100 groups (P < 0.05), while the expression level of CyclinB1 did not differ among the groups. The results indicated that a 50% concentration of GCCM in combination with BM components enhanced MII and viability rates and mitochondria activity of mouse immature oocytes.

Oocyte Vitrification Reduces its Capability to Repair Sperm DNA Fragmentation and Impairs Embryonic Development.

Oocytes play a crucial role in repairing sperm DNA damage, which can affect the next generation; however, certain factors can impair this ability. This study examined whether oocyte vitrification, a widely used method for fertility preservation, negatively affects repair ability. Male DBA/2 mice (n = 28) were injected with 101.60 µmol/100 g body weight of tert-Butyl hydroperoxide (tBHP) for 14 days to induce sperm DNA damage. Histological changes, sperm functions, and DNA fragmentation were assessed using the TUNEL assay. Cumulus-oocyte-complexes (COCs) of superovulated female DBA/2 mice (n = 28) were vitrified using the Cryotop method. Fresh and vitrified oocytes were then fertilized by tBHP-treated and untreated sperms, and subsequent embryonic development was monitored. Additionally, the expression of Mre11a, Rad51, Brca1, and Xrcc4 was assessed in resulting zygotes and blastocysts using real-time PCR. The sperm tBHP treatment reduced differentiated spermatogenic cells in the testicular tissue, sperm concentration, and motility, while increasing DNA fragmentation (P < 0.05). The fertilization rate was decreased in the tBHP-treated sperm-vitrified oocyte group (P < 0.05), and the two-cell rate diminished in tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05). The four-cell to blastocyst rate decreased in the untreated sperm-vitrified oocyte and the tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05), and the tBHP-treated sperm-vitrified oocyte groups had the lowest blastocyst rate. In zygotes, Brca1 was upregulated in the tBHP-treated sperm-vitrified oocyte group (P < 0.05). Also, in blastocysts, Rad51, Brca1, and Xrcc4 were significantly upregulated in the untreated sperm-vitrified oocytes group (P < 0.05). Damages to the oocyte due to vitrification can disrupt the repair of sperm DNA fragmentation and consequently impair the embryo development.

Simple bioelectrical microsensor: oocyte quality prediction via membrane electrophysiological characterization.

Oocyte selection is a crucial step of assisted reproductive treatment. The most common approach relies on the embryologist experience which is inevitably prone to human error. One potential approach could be the use of an electrical-based approach as an ameliorative alternative. Here, we developed a simple electrical microsensor to characterize mouse oocytes. The sensor is designed similarly to embryo culture dishes and is familiar to embryologists. Different microelectrode models were simulated for oocyte cells and a more sensitive model was determined. The final microsensor was fabricated. A differential measuring technique was proposed based on the cell presence/absence. We predicted oocyte quality by using three electrical characteristics, oocyte radii, and zona thicknesses, and also these predictions were compared with an embryologist evaluation. The evaluation of the oocyte membrane capacitance, as an electrophysiological characteristic, was found to be a more reliable method for predicting oocytes with fertilization and blastocyst formation success competence. It achieved 94% and 58% prediction accuracies, respectively, surpassing other methods and yielding lower errors. This groundbreaking research represents the first of its kind in this field and we hope that this will be a step towards improving the accuracy of the treatment.

Optimal human ovarian follicle isolation: A review focused on enzymatic digestion.

The damage or depletion of ovarian reserves due to aging or cancer treatment can increase the need for fertility preservation techniques. One of the most common ways of supporting fertility in prepubertal girls and women who require immediate cancer treatment is through ovarian tissue cryopreservation and re-transplantation following cancer treatment. However, a more appropriate method should be employed in diseases such as leukemia, where malignant cells may be present in cryopreserved tissue, instead of ovarian tissue transplantation. Human ovarian follicle isolation for in vitro culture or the use of artificial ovaries for their growth can decrease the risk of reintroducing cancer cells into these individuals. Here we review the methods for the isolation of human ovarian follicles.

Effect of Moderate Static Magnetic Fields on Mice Oocyte Vitrification: Calcium-Related Genes Expression.

The ability to cryopreserve oocytes without ultrastructural injury has been a concern in the development and use of methods to preserve female reproduction. The stability of the cell membrane must be preserved to reduce the damage caused by ice crystals during vitrification. One approach that has been explored is the use of static magnetic fields (SMFs), which are believed to influence cell membrane stability. In this study, the in vitro effects of SMF that range between 20-80 mT on the vitrification of mice germinal vesicle (GV) oocytes were studied. The viability and mitochondrial (Mt) membrane potential of both vitrified and nonvitrified oocytes were assessed using Trypan blue and JC1 staining. The high in vitro maturation (IVM) rate and high Mt membrane potential in metaphase II (MII) oocytes were taken into account to determine the optimal magnetic field intensity, that is, 20 mT. None of the SMF conditions resulted in intact spindles in MII oocytes. The study also explored the expression of store-operated calcium entry (Stim1, Orai1, and Ip3r) and meiosis resumption (Ccnb, Cdk) genes in GV and MII oocytes of both vitrified and control groups. The results show that the expressions of Orai1 and Ccnb genes in Vit-MII-SMF oocytes were considerably increased. However, no significant difference in Stim1 expression was observed between the groups. The Vit-MII-SMF group exhibited a significantly higher Ccnb expression compared to other groups. In vitro fertilization (IVF) was performed to evaluate the 2 pronuclear (2PN) rates. The findings demonstrated that using 20 mT SMF improved 2PN rates compared to the nonvitrified groups. This study provides a deeper understanding of the effects of moderate SMF and vitrification on the expression of calcium channel genes in GV and MII oocytes. The results suggest that applying a 20 mT SMF can help prevent cryoinjury and enhance the characteristics of vitrified-warmed oocytes.

Improvement of Mouse Preantral Follicle Survival and Development following Co-Culture with Ovarian Parenchyma Cell Suspension.

BACKGROUND: The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro. MATERIALS AND METHODS: In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods. RESULTS: The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions. CONCLUSION: The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

In-vivo oogenesis of oogonial and mesenchymal stem cells seeded in transplanted ovarian extracellular matrix.

OBJECTIVE (S): One way to overcome the recurrence of cancer cells following ovarian tissue transplantation is to use decellularized tissues as a scaffold that does not have any cellular components. These cell-free scaffolds can be seeded with different type of stem cells for ovarian restoration. MATERIALS AND METHODS: OSCs, PMSCs and BMSCs (oogonial, peritoneal and bone marrow mesenchymal stem cells, respectively) were seeded into human decellularized ovarian tissue as 4 groups: Scaffold + OSCs (SO), Scaffold + OSC + PMSCs (SOP), Scaffold + OSC + BMSCs (SOB) and Scaffold + OSC + PMSCs + BMSCs (SOPB). The produced grafts were transplanted into the sub-peritoneal space of ovariectomized NMRI mice as artificial ovary (AO). The expression of Vegf, CD34, Gdf9, Zp3, Ddx4, Amh and Lhr genes in AOs were measured by qRT-PCR. Also, histotechniques were considered to detect the anti GFP, PCNA, VEGF, GDF9, ZP3 and AMH proteins. RESULTS: H & E staining showed follicle-like structures in all groups; the number of these structures, in the SOP and SOB groups, were the highest. In SO group, differentiation ability to oocyte and granulosa cells was observed. Endothelial, oocyte, germ, and granulosa cell-like cells were specially seen in SOP and angiogenesis capability was more in SOB group. However, angiogenesis ability and differentiation to theca cell-like cells were more often in SOPB group. While none of the groups showed a significant difference in AMH level, estradiol levels were significantly higher in SOPB group. CONCLUSION: Integration of OSCs + PMSCs and those OSCs + BMSCs were more conducive to oogenesis.

Successful 3D culture and transplantation of mouse isolated preantral follicles in hydrogel of bioengineered Wharton's jelly.

MAIN OBJECTIVE: Due to Human Wharton's Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles. MATERIALS AND METHODS: HWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton's Jelly (DWJ) was dissolved to make Wharton's Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10µL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation. RESULTS: Collagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05). CONCLUSION: Wharton's jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.

Follicular reconstruction and neo-oogenesis in xenotransplantation of human ovarian isolated cells derived from chemotherapy-induced POF patients.

BACKGROUND: Developing new strategies to restore fertility in patients with chemotherapy-induced Premature Ovarian Failure (Chemo-POF) is important. We aimed to construct an Artificial Ovary (AO) by seeding Human Ovarian Cortical Cells (HOCCs) into Human ovarian Decellularized Cortical Tissue (DCT). We assessed the AO's ability to produce new ovarian follicles following xenotransplantation to NMRI mice. MATERIAL AND METHODS: The DCTs were prepared, and cell removal was confirmed through DNA content, MTT assay, DAPI and H&E staining. Next, HOCCs were isolated from both Chemo-POF and Trans (as a control group) ovarian patients. The HOCCs were characterized using immunostaining (FRAGILIS, Vimentin, and Inhibin α) and real time PCR (DDX4, STELLA, FRAGILIS, Vimentin, FSH-R, KI67) assays. The HOCCs were then seeded into the DCTs and cultured for one week to construct an AO, which was subsequently xenotransplanted into the mice. The existence of active follicles within the AO was studied with H&E and immunofluorescence (GDF9) staining, Real-time PCR (GDF9, ZP3) and hormone analysis (Estradiol, FSH and AMH). RESULTS: The results of gene expression and immunostaining showed that 85-86% of the isolated cells from both Trans and Chemo-POF groups were positive for vimentin, while 2-5% were granulosa cells and OSCs were less than 3%. After xenotransplantation, histological study confirmed the presence of morphologically healthy reconstructed human ovarian follicles. Additionally, immunofluorescence staining of GDF9 and hormonal assay confirmed the presence of secretory-active follicles on the AO. CONCLUSION: Our findings demonstrate that an artificial ovary produced by seeding HOCCs on DCT can support HOCCs proliferation as well as neo-oogenesis, and enable sex hormone secretion following xenotransplantation.

Development of 4-cell mouse embryos after re-vitrification.

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts.

Mouse ovarian follicle growth in an amniotic membrane-based hydrogel.

Three-dimensional cultures of follicles on ECM-based scaffolds can be an approach for women who become infertile after cancer treatments. Human amniotic membrane (HAM) is extensively employed in tissue engineering because of its unique properties. We cultured mouse pre-antral follicles in a hydrogel derived from decellularized amniotic membrane (DAM) combined with alginate (ALG) to improve ovarian follicle culture. HAM was decellularized. Quantitative (nuclear contents, collagen, glycosaminoglycan [GAG]) and qualitative (DAPI, H&E, Masson's trichrome, Alcian blue, scanning electron microscopy assessments were performed. Then, we created an amniotic membrane-based hydrogel (AMBH) and conducted AMBH characterization assays (rheology, MTS, degradation rate). Isolated mouse pre-antral follicles were cultured in 15 mg/mL AMBH (AMBH15), 30 mg/mL AMBH (AMBH30), or 45 mg/mL AMBH (AMBH45). ALG hydrogel was the control group. Follicular diameters, estradiol hormone secretion rate, follicular morphology, and the follicle antral and degeneration rate were examined. Quantitative and qualitative assays indicated successful decellularization. AMBH characterization assays showed that the ALG hydrogel had more appropriate gelation and slower degradation than AMBH. There was a statistically higher antral follicle formation rate in the AMBH45 group (p < .05) compared to the AMBH30 and AMBH15 groups and less (p < .05) degenerated follicles. There was no significant difference with the ALG group. Diameter and estradiol hormone secretion in the AMBH45 group were not significantly higher than the ALG group. Although decellularization was confirmed and the viscoelastic parameters of AMBH support follicle culture, there was no significant effect on ovarian follicle maturation compared to the ALG control group.