Transplantation of rat frozen-thawed testicular tissues: Does fragment size matter?

Cryopreservation of testicular tissue from pre-pubertal boys before gonadotoxic treatment is an important step in fertility preservation. Yet, this approach remains experimental, and there is still few study measuring the effect of tissue size on the graft after cryopreservation and transplantation. The objective of this study is to detect the effect of varying tissue sizes on the efficacy of rat testicular tissue cryopreservation and transplantation. Varying sizes of rat testicular tissues were frozen-thawed and autografted. At the 30th day after grafting, the grafts were collected for histology assessment and immunohistochemistry assay for MAGE-A4 (germ cell marker) and CD34 (blood vessel marker). The transplant recovery, seminiferous tubule integrity, tubular diameter, spermatogonia number, and microsvessel density in testicular fragments sizing in 3 mm in length, 3 mm wide, and 3 mm in thickness were significantly lower than other groups. Whereas, the absorption rate of graft sizing in 1 mm in length, 1 mm in wide, and 1 mm in thickness was significantly higher than other groups. Testicular fragment sizing in 2-3 mm in length, 2-3 mm in wide, and 2 mm in thickness (8 mm3-18 mm3) is suitable for rat testicular tissue cryopreservation and transplantation.

Targeting against the activity of the NLRP3 inflammasome is a potential therapy for rat testicular tissue cryopreservation and transplantation.

The objective of the present experiment was to explore the role of NLRP3 inflammasome in the testicular tissue freezing, thawing and grafting; furthermore, the potential effect of a NLRP3 inhibitor on the function of testis transplant was explored. Tissues from male Wistar rats in pre-pubertal age were cryopreserved, thawed and auto-transplanted into the scrotum treated or not treated with the MCC950 (a NLRP3 inhibitor). After grafting, cryopreserved tissue was removed and analysed. Quantitative morphometric, immunohistochemical techniques and Western blotting were used to evaluate the survival of spermatogonia and the activation of the NLRP3 inflammasome after freezing/thawing/grafting. Moreover, serum IL-1ß level was assessed with ELISA kits. The testicular transplants exhibited upregulated expression of the NLRP3 pathway meditors (NLRP3, IL-1ß). In NLRP3 inhibition group, the rate of recovered grafts, the percentage of intact tubules and spermatogonial number were significantly higher than that in cryopreserved graft group. Moreover, serum concentration of IL-1ß in NLRP3 inhibition group was significantly lower than that in cryopreserved graft group. Testicular tissue cryopreservation and transplantation exhibited upregulated expression of NLRP3 pathway and NLRP3 inflammasome blockade improves testicular graft function. These finding suggest that NLRP3 inflammasome is a therapeutic target for testicular tissue cryopreservation and transplantation.

Testicular damage during cryopreservation and transplantation.

The aim of this study is to do a study of cryoinjury and ischaemic injury on testicular graft during cryopreservation and transplantation. According to time at 1, 3, 7 and 14 days after transplantation, the grafts were collected for immunohistochemistry assay for CD34 (blood vessel marker), VEGF (neoangiogenesis marker), caspase-3 (apoptosis marker) MAGE-A4 (germ cell marker). A significant increase was observed in the density of VEGF-positive blood vessels on day 3, reached a peak on day 7. On post-transplant day 3, a sharp increase occurred in the rate of spermatogonia-expressing caspase-3 until the day 7. At 14th day after transplantation, the spermatogonia number per round tubule of nonfrozen grafts was 41 ± 5.9% from that of fresh control tissues, while, in frozen-thawed grafts, the spermatogonia number per round tubule was 36.8 ± 4.6% from that of fresh control tissues. In testicular grafts, angiogenesis initiated reperfusion from day 3, and the formation of new blood vessel generally is completed about 7 days after transplantation. Angiogenesis in grafts after transplantation plays a crucial role in the restoration of function. Therefore, minimising ischaemic injury as well as improvement of cryopreservation protocols are needed to improve testicular graft after freezing, thawing and grafting.

Effects of folic acid on oligozoospermia with MTHFR polymorphisms in term of seminal parameters, DNA fragmentation, and live birth rate: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: It has been reported that paternal folic acid deficiency is correlated with male infertility and increased birth defects in the offspring. However, there are few data concerning the influence of folic acid supplementation on male-factor infertility with MTHFR gene polymorphisms. OBJECTIVES: To evaluate whether folic acid supplementation has a beneficial effect on oligozoospermia with MTHFR gene polymorphisms in Chinese infertility population. MATERIALS AND METHODS: The infertile men suffering oligozoospermia with MTHFR gene polymorphisms were randomly divided into the folic acid treatment groups receiving folic acid 0.8 mg daily for 3 months and the placebo groups receiving placebo for 3 months. Semen parameters, seminal MDA, and DNA fragmentation were measured. Furthermore, spontaneous pregnancy rate and live birth rate were evaluated. RESULTS: Administration of folic acid for 3 months could significantly improve the seminal parameters in patients with MTHFR 677 TT genotype in comparison with that receiving placebo. Moreover, seminal MDA and sperm DNA fragmentation index in patients with MTHFR 677 TT genotype significantly declined at the end of treatment. Spontaneous pregnancy rate and live birth rate tended to be significantly higher in couples in which the men with MTHFR 677 TT genotype receiving folic acid than that receiving placebo. However, folic acid treatment did not exhibit any advantage in MTHFR 677 CT, 1298 AC, 1298 CC, 1793 GA, or combined 677 CT/1298 AC genotype. DISCUSSION: The anti-oxidation function of folic acid is one of possible mechanisms invovled in improving seminal parameters and pregnancy outcome. CONCLUSIONS: Folic acid supplementation has a beneficial effect on oligozoospermia with MTHFR 677 TT genotype in term of seminal parameters, seminal MDA, sperm DNA fragmentation, and pregnancy outcome.

Pyroptosis is involved in cryopreservation and auto-transplantation of mouse ovarian tissues and pyroptosis inhibition improves ovarian graft function.

The aim of this study was to investigate the role of pyroptosis in the cryopreservation and transplantation of mouse ovarian tissues; the effects of pyroptosis inhibitior on the ovarian graft function were also explored. ICR (institute of cancer research) mice were randomly divided into control group and experimental groups (n = 10 per group). The experimental groups included fresh graft group (autograft), cryopreserved graft group (cryopreservation + autograft), and pyroptosis inhibition group (cryopreservation + autograft + pyroptosis inhibitor). At the third day after auto-transplantation, caspase-1 and NLRP3 protein expression in grafts were assessed by Western blot; in the meantime, serum concentration of IL-1ß was examined by ELISA. After 28 days of auto-transplantation, estradiol concentrations and follicular densities of grafts were evaluated. The caspase-1 and NLRP3 protein expression in grafts from all the experimental groups were significantly higher than that from control group respectively; moreover, there was a significant increase in serum concentrations of IL-1ß in all experimental groups compared with control group. The concentration of estradiol and follicular densities of grafts in pyroptosis inhibition group were significantly higher than that in cryopreserved graft group. Pyroptosis is involved in cryopreservation and auto-transplantation of mouse ovarian tissues, and pyroptosis inhibition can improve the ovarian graft function.

Treatment of idiopathic oligozoospermia with combined human chorionic gonadotropin/human menopausal gonadotrophin: A randomised, double-blinded, placebo-controlled clinical study.

To evaluate whether hCG/hMG therapy has beneficial effects on idiopathic oligozoospermia in Chinese infertility population. The patients were randomly divided into the treatment group receiving hCG/hMG for 3 months and the placebo group receiving placebo for 3 months. Semen and biochemical analysis was performed, and DNA fragmentation as well as spermatid concentration was evaluated. Administration of hCG/hMG for 3 months could significantly improve sperm concentration, rate of forward motile spermatozoa, total motile sperm count, the percentage of sperm with normal morphology and the rate of spontaneous pregnancy in medium- and higher-level inhibin B group respectively. Moreover, in medium- and higher-level inhibin B group, sperm DNA fragmentation index and spermatid concentration were significantly declined respectively at the end of treatment. However, there were no significant differences in lower-level inhibin B group before and after treatment in term of seminal parameters, DNA fragmentation and spermatid concentration. HCG/hMG therapy for 3 months has a beneficial effect on a part of male with idiopathic oligozoospermia, and the efficacy of hCG/hMG therapy is associated with the inhibin B level.

Superovulation does not affect the endocrine activity nor increase susceptibility to carcinogenesis of uterine and mammary glands of female offspring in mice.

PURPOSE: To evaluate the dual effects of superovulation on the endocrine activity and susceptibility to carcinogenesis of uterine and mammary glands of female offspring in mice METHOD: The mice were superovaluted. The relative uterine weight, ERα protein expression, and endocrine activity of female offspring (F1 generation and F2 generation) were measured. Furthermore, proliferative lesion of uterine and mammary glands of female offspring (F1 generation and F2 generation) was assessed by histopathologic examinations. RESULTS: There were no significant differences in relative uterine weight, ERα protein expression, incidence of proliferative lesion in mammary glands, and incidence of atypical hyperplasia, adenocarcinoma, and squamous metaplasia in uterine among the offspring (F1 generation and F2 generation) in each group. Likewise, there were no significant intergroup differences in the serum levels of sex related hormones. CONCLUSIONS: No significant alterations were found in the endocrine activity and susceptibility to carcinogenesis of uterine and mammary glands of female offspring in mice produced by superovaluted oocytes compared with those of naturally conceived offspring.

Effects of varying tissue sizes on the efficiency of baboon ovarian tissue vitrification.

OBJECTIVE: The aim of this study was to detect the effects of varying tissue sizes on the efficiency of baboon ovarian tissue vitrification. STUDY DESIGN: The percentages of morphologically normal primordial follicles and the follicles expressing bax protein in ovarian tissues after vitrification-warming were measured. Besides, the 17-ß estradiol levels in the culture supernatants were measured. RESULTS: The percentages of morphologically normal primordial follicles in vitrified-warmed ovarian tissues slicing in 0.5-1.5mm in length and wide, and 1.0mm in thickness were significantly higher than those slicing in 2.0mm in length and wide, and 1.0mm in thickness. Moreover, the follicles expressing bax protein in vitrified-warmed ovarian tissues slicing in 0.5-1.5mm in length and wide, and 1.0mm in thickness were significantly lower than those slicing in 2.0mm in length and wide, and 1.0mm in thickness. The 17-ß estradiol levels in the culture supernatants slicing in 1.0-1.5mm in length and wide, and 1.0mm in thickness were significantly higher than those slicing in 0.5mm or 2.0mm in length and wide, and 1.0mm in thickness. CONCLUSIONS: Cortex piece slicing in 1.0-1.5mm in length and wide, and 1.0mm in thickness is suitable for baboon ovarian vitrification.

The long-term effects of superovulation on fertility and sexual behavior of male offspring in mice.

PURPOSE: To evaluate the long-term effects of superovulation on fertility and sexual behavior of male offspring in mice. METHOD: The mice were superovaluted, and the fertility of male offspring (F1 generation and F2 generation) were evaluated in terms of the percentage of plugs and pregnancies, serum testosterone concentrations, and sperm motility. Furthermore, the sexual behavior of male offspring and sex ratio (F1 generation and F2 generation) were measured. RESULTS: There were no significant differences in the percentage of plug and pregnancies, serum testosterone concentrations, sperm motilities and sex ratio between the offspring in naturally conceived group and superovulation groups (both F1 generation and F2 generation). The sperm hyperactivity at 90 min after incubation of F1 generation in naturally conceived group were higher than that of F1 generation in superovulation group, but the differences did not reach statistical significance. The offspring produced by superovaluted oocytes (both F1 generation and F2 generation) did not exhibit significant alterations in sexual behavior. CONCLUSIONS: No significant alterations were found in fertility and sexual behavior of male offspring in mice produced by superovaluted oocytes compared with those of naturally conceived offspring.

In vitro maturation of oocytes is not a risk factor for adult metabolic syndrome of mouse offspring.

OBJECTIVE: To evaluate the effect of in vitro maturation (IVM) of oocytes on the serum cholesterol profile, glucose and insulin tolerance, blood pressure, and heart rate of adult mouse offspring. STUDY DESIGN: The serum cholesterol profile, glucose and insulin tolerance, blood pressure, and heart rate of adult offspring born from in vitro matured oocytes were compared with those of adult offspring born from in vivo matured oocytes. RESULTS: Offspring born from in vitro matured oocytes showed a normal serum cholesterol profile. In the glucose tolerance test, glucose levels were consistently elevated in adult offspring born from in vitro matured oocytes compared with those born from in vivo matured oocytes, but the differences did not reach statistical significance. There were no significant differences in insulin tolerance, blood pressure, and heart rate between adult offspring born from in vitro matured oocytes and those from in vivo matured oocytes. CONCLUSION: No alterations were found in the metabolism profile of adult mouse offspring born from in vitro matured oocytes compared with that from in vivo matured oocytes.

Effect of vitrification of mouse oocyte on the behavior of adult offspring.

OBJECTIVE: To evaluate the effect of vitrification of mouse oocytes on the behavior of adult offspring. STUDY DESIGN: Oocytes from mice were vitrified, warmed and inseminated, and two-cell embryos were transferred to foster mothers. The behavioral characterization of the offspring was detected by the Morris water maze test, forced swimming test, and elevated plus maze test, and compared to that of offspring from fresh oocytes. RESULTS: Offspring produced by vitrified oocytes showed normal motor function. In the Morris water maze test of spatial learning there was a slightly decreased time spent in the quadrant containing the platform relative to mice from fresh oocytes, but the difference did not reach statistical significance. In addition, offspring from vitrified oocytes did not exhibit alterations in emotional behavior. CONCLUSION: No alterations were found in the behavioral characterization of adult offspring from vitrified oocytes compared with those from fresh oocytes.