Preservation Of Germ Cells Research Articles

Damages of Freezing on the Development of Oocyte and Embryos

Introduction: To assist save endangered species from extinction and to aid in their care, it is crucial to sustaining their reproductive capacity. The fetus's life and growth processes begin at conception and proceed in accordance with a biological clock's timing. With today's understanding through cryobiology, observing scientific ethics problems and interfering with the clock's operation by stopping its biological time is possible. Practically, it is accomplished by storing the cell at -196 °C, or the temperature of liquid nitrogen, where all metabolic activity ceases. Since 200 years ago, germ cell preservation has been a common practice. Since then, there have been many improvements, particularly for at-risk women. Since then, significant progress has been achieved, and several freezing techniques are being used to preserve ovarian tissue, follicles, and oocytes in women who are at risk of infertility. Different approaches have different levels of success. Among preservation techniques, vitrification performs better and is used more frequently. The cellular configuration of the mammalian oocyte is intricate. This cell's constituent parts are particularly sensitive to osmosis and variations in temperature. For instance, the alterations to the cell membrane that occur during maturation, in vitro fertilization, and the differential between the permeability of water and cryoprotectants can all be mentioned. Oocyte freezing results in a variety of impairments, including a reduction in the quality and viability of cells after thawing. Recent studies are looking for ways to enhance freezing procedures and raise the caliber of frozen oocytes. The favorable or negative effects of freezing on the oocyte and its potential, or embryo development, are the subject of this review article.

Folic Acid Preconditioning Alleviated Radiation-Induced Ovarian Dysfunction in Female Mice.

Radiological therapy/examination is the primary source of artificial radiation exposure in humans. While its application has contributed to major advances in disease diagnosis and treatment, ionizing radiation exposure is associated with ovarian damage. The use of natural products, either alone or as an adjunct, has become increasingly common for reducing the side effects of radiological therapy during disease treatment. Herein, we explored the protective effect of folic acid (FA), a widely used B vitamin, against radiation-induced ovarian injury and its mechanism of action. Female mice with normal ovarian function were randomly divided into control, FA, radiation, and radiation + FA groups. The intervention strategy included daily intragastric administration of FA (5 mg/kg) for 3 weeks prior to radiation exposure. Mice in the radiation and radiation + FA groups received a single dose of 5 Gy X-ray irradiation. Changes in the estrous cycle were then recorded, and ovarian tissues were collected. Pathophysiological changes as well as reproductive and endocrine-related indexes were determined via H&E staining, immunohistochemistry, Western blot, and ELISA. The reproductive performance and emotional symptoms of animals were also monitored. Our results indicated that FA intervention effectively alleviated ovarian damage, leading to more regular estrous cycles, lesser impairment of follicular morphology and endocrine status, as well as greater germ cell preservation. Reduced levels of oxidative stress, inflammation, and enhanced DNA repair were associated these changes. FA pre-administration improved the reproductive performance, leading to higher pregnancy rates and greater litter sizes. Further, the anxiety levels of animals were significantly reduced. Our results indicate that FA pre-administration significantly alleviates radiation-induced ovarian damage in rodents, highlighting its potential as a protective strategy against radiation exposure in the female population.

Long-term preservation of germ cells and gonadal tissues at ambient temperatures.

ObjectiveTo present an overview of different approaches and recent advances for long-term preservation of germ cells and gonadal tissues at ambient temperatures.MethodsReview of the existing literature.ResultsPreserving viable spermatozoa, eggs, embryos, and gonadal tissues for the long term is critical in human fertility treatment and for the management of animal populations (livestock, biomedical models, and wild species). The need and number of banked germplasms are growing very fast in all disciplines, but current storage options at freezing temperatures are often constraining and not always sustainable. Recent research indicates that structures and functions of gametes or gonadal tissues can be preserved for the long term using different strategies based on dehydration and storage at supra-zero temperatures. However, more studies are needed in rehydration and reanimation of germplasms (including proper molecular and cellular evaluations).ConclusionsWhile a lot of research is still warranted to optimize drying and rehydration conditions for each sample type and each species, alternative preservation methods will change the paradigm in fertility preservation and biobanking. It will transform the way we maintain and manage precious biomaterials for the long term.Lay summaryLiving sperm cells, eggs, embryos, and reproductive tissues can be preserved at freezing temperatures for human fertility treatments and used to manage breeding in livestock, laboratory animals, and wild species through assisted reproduction. These cells can be stored in cell banks and demand for them is growing fast. However, current long-term storage options at freezing temperatures are expensive. Instead of using low temperatures, recent research indicates that these cells can be dried and stored above freezing temperatures for an extended amount of time. While a lot of research is still needed to optimize how different samples are dried and rehydrated, alternative methods of preserving cells will make fertility preservation and cell banking easier. It will also transform the way we keep and manage samples for the long term.

Effect of miR-302b MicroRNA Inhibition on Chicken Primordial Germ Cell Proliferation and Apoptosis Rate.

The primordial germ cells (PGCs) are the precursors for both the oocytes and spermatogonia. Recently, a novel culture system was established for chicken PGCs, isolated from embryonic blood. The possibility of PGC long-term cultivation issues a new advance in germ cell preservation, biotechnology, and cell biology. We investigated the consequence of gga-miR-302b-5P (5P), gga-miR-302b-3P (3P) and dual inhibition (5P/3P) in two male and two female chicken PGC lines. In treated and control cell cultures, the cell number was calculated every four hours for three days by the XLS Imaging system. Comparing the cell number of control and treated lines on the first day, we found that male lines had a higher proliferation rate independently from the treatments. Compared to the untreated ones, the proliferation rate and the number of apoptotic cells were considerably reduced at gga-miR-302b-5P inhibition in all PGC lines on the third day of the cultivation. The control PGC lines showed a significantly higher proliferation rate than 3P inhibited lines on Day 3 in all PGC lines. Dual inhibition of gga-miR-302b mature miRNAs caused a slight reduction in proliferation rate, but the number of apoptotic cells increased dramatically. The information gathered by examining the factors affecting cell proliferation of PGCs can lead to new data in stem cell biology.

Effects of Estrogen on Spermatogenesis in Transgender Women

ObjectiveTo characterize spermatogenesis in the estrogenized transgender patient. Materials and MethodsThis is a retrospective, single-center, cross-sectional study. Seventy-two transgender women underwent gender-affirming orchiectomy between May 2015 and January 2017. All were on long-term (>1 year) cross-sex hormonal therapy prior to orchiectomy. Patient data were obtained via chart review. Histologic analysis was performed by a pathology resident under the supervision of a genitourinary pathologist. The main outcome is histologic presence of germ cells and presence of spermatids (a proxy for preserved spermatogenesis) in orchiectomy specimens. ResultsThere were 141 pathologic specimens available for analysis. Germ cells were present in 114 out of 141 (81%) testicles. Spermatids were present in 57 (40%) testicles. Presence of germ cells was associated with older age (43 vs 35 years, P = .007) and increased testicular weight (28.6 g vs 19.3 g, P <.001). Presence of spermatids was associated with increased weight (31.5 g vs 23.3 g, P <.001) and volume (20.3 mL vs 12.6 mL, P <.001). There was a linear correlation between testis volume and preserved spermatogenesis (Pearson's r = 0.448, P <.001). ConclusionDespite long-term hormone therapy, the majority (80%) of transgender women have germ cells present in the testicle. Spermatogenesis is preserved in approximately 40% of these individuals. Duration of hormonal therapy did not affect the degree of preservation of germ cells or spermatogenesis but starting hormonal treatment at a younger age may be associated with decreased germ cells in the testicle. Volume of testicles predict presence of preserved spermatogenesis.

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration.

During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA-sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.

Presence of Germ Cells in Disorders of Sex Development: Implications for Fertility Potential and Preservation

We sought to determine the presence of germ cells in the gonads of patients with disorders of sex development to establish whether preservation of germ cells for future fertility potential is possible. We hypothesized that germ cells are present but vary by age and diagnosis. We reviewed histology from patients with disorders of sex development who underwent gonadectomy/biopsy from 2002 to 2014 at a single institution for pathological classification of the gonad, composition of gonadal stroma and germ cell presence. A total of 44 patients were identified and germ cells were present in 68%. The presence and average number of germ cells per mm2 were analyzed by gonad type and diagnosis. By gonad type all ovotestes, most testes, ovaries and dysgenetic testes, and 15% of streak gonads had germ cells present. By diagnosis germ cells were present in all patients with complete androgen insensitivity syndrome, Denys-Drash syndrome, SRY mutation, mixed gonadal dysgenesis, ovotesticular conditions and StAR (steroid acute regulatory protein) deficiency, in some patients with persistent müllerian duct syndrome, XO/XY Turner syndrome and disorders of sex development not otherwise specified, and in none with complete or partial gonadal dysgenesis. Germ cells were present in the gonads of 88% of patients 0 to 3 years old, 50% of those 4 to 11 years old and 43%of those older than 12 years. Germ cells were present in the majority of our cohort and the presence decreased with age. This novel, fertility driven evaluation of germ cell quantity in a variety of disorders of sex development suggests that fertility potential may be greater than previously thought. Further studies must be done to evaluate a larger population and examine germ cell quality to determine the viability of these germ cells.

Carnitine partially protects the rat testis against the late damage produced by doxorubicin administered during pre‐puberty

Doxorubicin, an anticancer drug, is widely included in chemotherapy protocols to combat childhood cancer. Carnitine, an important quaternary amine, is present in testis and epididymis and is involved in sperm maturation; it has been used in infertility treatment. In a previous study, our group observed that L-carnitine given before etoposide, another chemotherapeutic drug, reduces the spermatogenic damage and protects germ cells against apoptosis. This study aimed to evaluate the antiapoptotic and cytoprotective actions of L-carnitine in long- and mid-term basis, on the seminiferous epithelium of doxorubicin-treated pre-pubertal rats. Forty-eight 30-day-old male Wistar rats were distributed into four groups: sham-control; doxorubicin; carnitine; carnitine/doxorubicin (L-carnitine injected 1 h before doxorubicin). The rats were submitted to euthanasia at 64 and 100 days of age and their testes were collected for biometric, morphometric, and histopathological analyses. The numerical density of apoptotic germ cells was obtained (TUNEL method). In adult phase (100 days), the following spermatic parameters were analyzed: mature spermatid (19 step) count and sperm daily production per testis; sperm number and transit time through the epididymal caput/corpus and cauda; frequency of morphologically abnormal spermatozoa (from epididymal fluid), as well as sperm DNA integrity (Comet assay). The testicular and spermatic parameters at both ages were improved in rats treated with carnitine before doxorubicin. At 64 days, the TUNEL-positive germ cell frequency was lower in the carnitine/doxorubicin-treated rats comparatively to the doxorubicin-treated rats. At 100 days of age, the sperm DNA fragmentation was also lower in the previously carnitine-treated rats, as evidenced by the analysis of three parameters. Carnitine reduced the late testicular and spermatic damages caused by doxorubicin, probably providing a partial cytoprotection against the deleterious action of doxorubicin administration to pre-pubertal rats. However, further studies shall be undertaken to investigate the protective mechanisms involved in such germ cell preservation.

From germ cell preservation to regenerative medicine: an exciting research career in biotechnology.

Collection, manipulation, assessment, and storage of mammalian gametes, embryos, and stem cells are providing important opportunities in agriculture, research, and medicine. Semen and embryo freezing in livestock are used in breeding schemes, especially in cattle and for international trade, with no risk of spreading disease. In human medicine, they are used in treatment of infertility. Usually, knowledge gained in one species is applicable in the others. In one exception, some ruminant embryos cultured according to protocols used in human in vitro fertilization become unusually large offspring. This is due to disturbances in expression of imprinted genes. The nuclear transfer procedure developed at the Roslin Institute is being used to make genetic modifications in livestock to either direct production of biomedical proteins, create animal models of human disease, or enhance animal health and productivity. Human pluripotent cells are being used in Edinburgh to identify drugs to treat degenerative diseases.

74 GERM CELLS AND TESTICULAR SOMATIC CELLS HAVE DIFFERENT SENSITIVITY TO CRYOPRESERVATION

Spermatogonial stem cells (SSC) are the foundation of spermatogenesis. Undifferentiated spermatogonia, containing SSC, represent only 2 to 5% of cells recovered from immature mammalian testis. Cryopreservation in liquid nitrogen allows for long-term storage of cells. Preservation of germ cells can serve as a means of genetic preservation from immature males when sperm storage is not an option. Studies have investigated the effects of cryopreservation on the spermatogenic potential of SSC and the efficiency of various cryopreservation protocols. Preliminary observations indicated that germ cells may survive cryopreservation better than testicular somatic cells, resulting in a post-thaw cell population enriched in germ cells. However, this has not been critically evaluated. The objective of this study was to test the hypothesis that germ cells are less susceptible to cryo-damage than testicular somatic cells. Cells were harvested from the testes of 1-wk-old piglets by 2-step enzymatic digestion. The initial cell suspension was subjected to differential adhesion to enrich the cell population for germ cells. Cells were plated in DMEM + 5% fetal bovine serum and incubated at 37°C in 5% CO2 in air. After 18 h, cells in suspension and cells slightly attached were recovered by trypsinization (1 : 10 trypsin-ethylenediaminetetraacetic acid) for 30 s and replated. This was repeated 24 and 36 h after initial plating. The enriched population was placed into cryovials at a concentration of 30 × 106 cells in freezing media (70% DMEM + 20% fetal bovine serum + 10% dimethyl sulfoxide), kept for 24 h at –80°C in a cryogenic freezing container and transferred to liquid nitrogen for 1 week. Aliquots of cells before freezing and after thawing at 37°C followed by incubation at 37°C in 5% CO2 in air for 1 h were analyzed for viability by propidium iodide (PI) exclusion and immunofluorescence for the germ cell marker VASA to identify viable germ cells (VASA+/PI–), nonviable germ cells (VASA+/PI+), viable somatic cells (VASA–/PI–), and nonviable somatic cells (VASA–/PI+). The percentage of viable germ cells after freezing and thawing was compared to the percentage of viable somatic cells by ANOVA. After enrichment by differential plating, the cell population had 95.6 ± 0.9% viability and contained 27.1 ± 7.4% germ cells (n = 3 replicates). After cryopreservation, the overall cell viability was 77.5 ± 1.6%, and 25.8 ± 8.0% were germ cells. The overall viability after cryopreservation could potentially have benefited from the 1-h incubation prior to analysis. The viability of the germ cell population after freezing and thawing was higher (92.1 ± 3.1%) than somatic cell viability (72.3 ± 1.7%; P &lt; 0.01). These results indicate that porcine germ cells survive cryopreservation better than do testicular somatic cells. Therefore, cryostorage of germ cells can be an efficient means for preservation of male genetic material. Supported by NIH ORIP/DCM RR17359.

Embryo transfer following in vitro maturation and cryopreservation of oocytes recovered from antral follicles during conservative surgery for ovarian cancer

IntroductionTherapeutic advances have significantly improved the prog-nosis of cancer patients [1]. This has generated new expect-ations especially for women of adolescent and reproductiveage for whom an increased hope of recovery from diseaseimplies a prospect of parenthood [2]. Unfortunately, radio-and chemotherapies have major effects on ovarian function,often leading to premature ovarian failure. Over the lastseveral years, different strategies have been developed topreserve female germ cells and, with them, reproductivefunction. Before a cancer treatment is started, parts of ovar-ian cortex can be explanted, cryopreserved and re-implantedorthotopically after clinical remission. With this approach,ovarian and reproductive function can be restored, at leasttransiently, as demonstrated by the birth of naturally con-ceived children [3]. Alternatively, following controlledovarian stimulation, mature oocytes can be retrieved, cryo-preserved and used at later stages to achieve a pregnancywith embryos generated in vitro [4]. Retrieval and storage ofimmature or in vitro matured oocytes may offer an addition-al opportunity for female germ cell preservation in cancerpatients [5]. In fact, germinal vesicle (GV)-stage oocytescan be collected from antral follicles in the absence ofgonadotropin administration and cryopreserved before orafter in vitro maturation (IVM). Therefore, IVM may bepreferable in cases in which tumour estrogen-sensitivityand/or urgency to start therapy conflict with the implemen-tation of a controlled ovarian stimulation treatment. In thisreport, we describe the recovery of immature oocytes fromantral follicles during a laparotomic conservative surgery forovarian cancer. These oocytes were matured in vitro, cry-opreserved by vitrification at the mature stage and subse-quently warmed to pursue a pregnancy in an IVF cycle. Thisexperience offers the proof of principle that opportunisticretrieval of immature oocytes during surgery is a realisticpossibility to preserve female reproductive potential.Case reportIn July 2010, a 38-year-old woman with a previous historyof infertility (one spontaneous abortion in 2005, three intra-uterine inseminations in 2008, and one ICSI cycle in April2010) underwent laparotomic surgery for ovarian adenocar-cinoma. The tumour displayed moderate differentiation(G2) and was staged as IIC according to the FIGO classifi-cation [6]. In the course of intervention, the extent andseverity of lesions required contextual right annexectomyand excision of an intact cyst from the left ovary.In consideration of the clinical condition and desire ofparenthood by the patient, and with approval of the local

Fertility of mice receiving vitrified adult mouse ovaries

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

Fertility and Pregnancy Outcome after Treatment for Cancer in Childhood or Adolescence

Successful therapy for children and adolescents with cancer includes the use of ionizing irradiation and/or chemotherapeutic agents. These may produce DNA damage, resulting in cell death, or the damage may be sublethal. These effects may be expressed in the gonads as sterilization or germ cell DNA damage. Sterilization may be acute, or identified by the occurrence of premature menopause. DNA damage may be identified by an increased risk for chromosomal syndromes, single gene defects or major congenital malformations in the offspring. Management of pediatric and adolescent cancer patients must include recognition of the potential for germ cell injury, counseling of patients regarding strategies for germ cell preservation, and long-term follow-up of the offspring of pediatric and adolescent cancer survivors to determine their increased risk, if any, for adverse pregnancy outcome, genetic disease and cancer.

Significance of epididymal and ductal anomalies associated with undescended testis: Study in 652 cases

Objective. We have long been interested in epididymal and ductal anomalies associated with cryptorchidism in the literature. We decided to study their prevalence in our cases due to the significance of these anomalies in patients' future fertility. Methods. We studied six hundred fifty-two testes treated for failed descent in our department and evaluated the epididymal and ductal anomalies we observed intraoperatively. Results. Epididymal and vasal anomalies occurred with an overall frequency of 36 percent (235 of 652 cases). Flimsy attachment of the head of epididymis to the testis constituted the most common anomaly. Conclusions. The prognosis of future fertility should consider the epididymal anomalies detected at orchiopexy. Such anomalies may coexist with excellent testis histology so that fertility impairment may occur based on sperm transport, despite the preservation of germ cells by early orchiopexy.

Significance of Epididymal and Ductal Anomalies Associated with Testicular Maldescent

Epididymal abnormalities have long been found in conjunction with cryptorchid testicles. The aim of this study was to document the different types of epididymal and ductal anomalies in relation to the position of the cryptorchid testis and to determine their clinical significance. Exploration was performed on 187 cryptorchid testes in 174 boys. The position of the testis and the epididymal and ductal anomalies were documented at operation. A biopsy was taken from the testis or atretic spermatic cord structures for light microscopy. Epididymal, ductal and/or testicular anomalies were detected in 43 per cent of the 187 testes, including anomalies of ductal fusion, anomalies of ductal suspension and anomalies associated with absent or vanishing testes. Biopsy of the testes with severe anomalies of ductal fusion showed preservation of germ cells in 69 per cent and diminished germ cells in 31 per cent. The higher the arrest of testicular descent, the more grossly abnormal was the associated ductal system. Early successful orchiopexy alone may not ensure subsequent fertility despite the presence of normal germ cells.