Adverse effects of ovarian cryopreservation and auto-transplantation on ovarian grafts and quality of produced oocytes in a mouse model.

The process of ovarian cryopreservation and transplantation is the only feasible fertility preservation method for prepubertal girls and female patients with cancer who cannot delay radiotherapy and chemotherapy. However, basic research on this technique is lacking. To better understand ovarian function and oocyte quality after ovarian tissue (OT) transplantation, we characterised the appearance, angiogenesis, and endocrine function of ovarian grafts in a murine model; the mitochondrial function and DNA damage in oocytes isolated from the OT; and the development of embryos after in vitro fertilisation. The results showed a decrease in oocyte numbers in the transplanted OT, abnormal endocrine function of ovarian grafts, as well as dysfunctional mitochondria and DNA damage in the oocytes, which could adversely affect subsequent embryonic development. However, these adverse phenotypes were partially or completely resolved within 21 days of transplantation, suggesting that ovulation induction and assisted pregnancy treatment should not be conducted too soon after OT transfer to ensure optimal patient and offspring outcomes.

Attitudes toward posthumous assisted reproduction in China: a multi-dimensional survey.

BACKGROUND: Professional legislation and ethics guidelines for posthumous assisted reproduction (PAR) are lacking in China. This study aims to measure the attitudes of the general public, IVF couples, and assisted reproductive technology (ART) practitioners toward PAR in China. METHODS: A multi-dimensional survey was designed, and electronic questionnaires were used. General demographic data, reproductive viewpoints, attitudes toward PAR, interactive ability to predict the partner's attitude toward PAR, and the legal attributes and rights to the disposal of posthumous embryos were evaluated. RESULTS: The study found that the traditional Chinese viewpoints of fertility had changed. The approval rates for PAR were 79.10%, 55.32%, and 58.89%, in the general public, IVF couples, and ART practitioners, respectively. Most participants agreed that the psychological well-being of offspring should be previously considered before making a PAR decision (81.84%, 73.61%, and 76.98%, respectively). Multivariable logistic regression analysis showed that age, marital status, and gender were common influencing factors, while occupation, religion, and pregnancy history showed no influence on support for PAR. Males and females showed similar predictive abilities for their partners' attitudes toward PAR (57.87% for males, 61.12% for females). Intracouple agreement analysis showed that the consistent rate of consistency in attitudes toward PAR was 65.28%. CONCLUSION: The findings suggested that the approval rate of PAR was relatively high in China. Legislation and ethics guidelines for PAR may be considered in China. The psychological well-being of offspring should be considered before the implementation of PAR. Due to the very large regional and demographic differences in China, investigation of a larger samples of participants is necessary.

This study is based on the dilemma of how to deal with the remaining frozen embryos when a family structure changes (such as the accidental death of one or both partners). In this research, we systematically investigated the basic attitudes of different groups toward PAR, the consistency and prediction accuracy of attitudes between couples and their ability to predict their partners' attitudes, and the balance between offspring well-being and reproduction through a multi-dimensional cross-sectional survey in China. Our study illustrated that the approval rates of PAR were relatively high among the public, IVF couples and ART practitioners. Couples' attitude prediction accuracy and the intercouple concordance were moderate. The psychological well-being of offspring should be considered before the implementation of PAR. Moreover, an appropriate legal policy or specialized guidance for PAR may be considered and published in China. This research provides some advice and evidence for medical professionals and policymakers regarding practice and policymaking related to PAR. We also believe that this manuscript is valuable and helpful for all the researchers who are interested in the posthumous reproduction, not only in China.

Effect of epigallocatechin-3-gallate (EGCG) on embryos inseminated with oxidative stress-induced DNA damage sperm.

Cryopreservation can induce damage in human spermatozoa through reactive oxygen species (ROS) generation. To reduce the potential risk of oxidative stress-induced sperm DNA damage, addition of different epigallocatechin-3-gallate (EGCG) concentrations were performed to determine the optimum concentration which was beneficial for IVF outcome for both fresh and frozen-thawed sperm. Next, the mouse sperm model exhibiting oxidative stress-induced DNA damage by exogenously treating with H2O2 but overcoming the low fertilization rate of frozen-thawed sperm was used to investigate the effect of EGCG on the embryonic development and the potential EGCG-mediated effects on ataxia telangiectasia mutated (ATM) pSer-1981 in zygotes, the latter was known for leading to the activation of major kinases involved in the DNA repair pathway and the cell cycle checkpoint pathway. We found the fertilization and embryonic development of embryos inseminated with frozen-thawed sperm was impaired compared to fresh sperm. EGCG promoted the development of embryos inseminated with both types of sperm at optimum concentration. In embryos inseminated with the H2O2 sperm, fertilization, embryonic development, and the time at which the cleavage rate of one-cell embryos reached ≥95% were not affected by EGCG treatment. However, the EGCG-treated group required less time to achieve 50% cleavage rate of one-cell embryos, and the EGCG-treated zygotes showed enhanced expression of ATM (pSer-1981) than the untreated group. EGCG at optimum concentrations may exert beneficial effects by modulating the ATM activation and moving up the time to enter into mitotic (M) phase. ABBREVIATIONS: ROS: reactive oxygen species; EGCG: epigallocatechin-3-gallate; ATM: ataxia telangiectasia mutated; M: mitotic.

Aurora B prevents aneuploidy via MAD2 during the first mitotic cleavage in oxidatively damaged embryos.

OBJECTIVES: A high rate of chromosome aneuploidy is exhibited in in vitro fertilization (IVF)-derived embryos. Our previous experiments suggested that reactive oxygen species (ROS) can activate Mad2, a key protein in the spindle assembly checkpoint (SAC), and delay the first mitotic, providing time to prevent the formation of embryonic aneuploidy. We aimed to determine whether mitotic kinase Aurora B was involved in the SAC function to prevent aneuploidy in IVF-derived embryos. MATERIALS AND METHODS: We analysed aneuploidy formation and repair during embryo pre-implantation via 4',6-diamidino-2-phenylindole (DAPI) staining and karyotype analysis. We assessed Aurora B activation by immunofluorescence and investigated the effect of Aurora B inhibition on embryo injury-related variables, such as embryonic development, ROS levels, mitochondrial membrane potential and γH2AX-positive expression. RESULTS: We observed the expression and phosphorylation of Thr232 in Aurora B in oxidative stress-induced zygotes. Moreover, inhibition of Aurora B caused chromosome mis-segregation, abnormal spindle structures, abnormal chromosome number and reduced expression of Mad2 in IVF embryos. Our results suggest that Aurora B causes mitotic arrest and participates in SAC via Mad2 and H3S10P, which is required for self-correction of aneuploidies. CONCLUSIONS: We demonstrate here that oxidative stress-induced DNA damage triggers Aurora B-mediated activation of SAC, which prevents aneuploidy at the first mitotic cleavage in early mouse IVF embryos.

CHK1-CENP B/MAD2 is associated with mild oxidative damage-induced sex chromosome aneuploidy of male mouse embryos during in vitro fertilization.

A high incidence of aneuploidy is observed in vitro fertilization (IVF)-derived embryos, but the formation and repair mechanisms are unknown. Here, we investigated the effects of slightly increased reactive oxygen species (ROS) produced by in vitro culture conditions on embryo aneuploidy and the roles of the spindle assembly checkpoint (SAC) protein, mitotic arrest-deficient 2 (MAD2), and the DNA damage response (DDR) protein, checkpoint kinase 1 (CHK1), in aneuploidy repair. By assessing chromosome abnormalities via DAPI staining, karyotype analysis and next-generation sequencing technology, we demonstrated that mild oxidative damage mainly increased the risk of sex chromosome aneuploidy in male mouse embryos (41,XXY,+X and 41,XYY,+Y) through chromosome mis-segregation during the first mitosis. Isobaric tags for relative and absolute quantitation technology revealed that mild oxidative damage inhibited the expression of male reproduction-related proteins, including a kinase anchor protein 4 (AKAP4), whose gene is located on mouse/human Chromosome X. Under mild oxidative damage, abrogation of MAD2 by MAD2 inhibitor-1 (M2I-1) or CHK1 by siRNA microinjection increased sex chromosome mosaicism rate and reduced mitosis-promoting factor (MPF) activity. CHK1 inhibition also reduced kinetochore localization of centromere protein B (CENP B) and MAD2. These findings show that DDR and SAC are responsible for repair of sex chromosome mosaicism via the pCHK1 (S345)-CENP B/MAD2-MPF pathway; further, IVF may have negative effects on male offspring's reproduction ability, which ultimately depends on their continued repair capability. Therefore, we suggest that antioxidants, especially those targeting improved CHK1-MAD2 function, may be a promising therapeutic strategy to reduce aneuploidy formation of IVF-derived embryos and to maintain genome integrity of embryo and offspring.

Oxidative Stress Delays Prometaphase/Metaphase of the First Cleavage in Mouse Zygotes via the MAD2L1-Mediated Spindle Assembly Checkpoint.

In zygotes, DNA damage delays the first cleavage to enable repair. Our previous study found that 0.03 mM hydrogen peroxide (H2O2) was the minimum concentration required for induction of oxidative DNA damage in mouse zygotes and that this represented the most similar situation to the clinical phenomenon. In this study, we quantified the cleavage rates of cells in blastocysts at different developmental stages, followed by immunofluorescence to detect activation of γ-H2A histone family member X (a marker of DNA damage) in zygotes to confirm that oxidative DNA damage was induced in H2O2-treated zygotes. Monitoring H3S10P (phosphorylation of Ser10 on histone H3; a prometaphase/metaphase marker) levels at different hour postinsemination revealed that treatment of zygotes with 0.03 mM H2O2 resulted in a prometaphase/metaphase delay. Furthermore, immunofluorescence staining for mitotic arrest deficient 2-like 1 and the protein kinase TTK, components of the spindle assembly checkpoint (SAC), suggested that this delay possibly involved SAC activation. These studies of the relationships between oxidative stress and SAC can promote the success rate of in vitro fertilization.

Effects of GnRH agonists on the expression of developmental follicular anti-mullerian hormone in varying follicular stages in cyclic mice in vivo.

Gonadotrophin­releasing hormone (GnRH) agonists (GnRHa) have been widely used to induce a state of downregulation for in vitro fertilization, and its direct effects on the pituitary are well known. However, the effects of GnRHa on the expression of anti­mullerian hormone (AMH) by follicles in varying stages in vivo remain to be fully elucidated. In the present study 84 cyclic mice were randomly divided equally into four GnRHa groups and three cyclic mice were used as a control group. The expression levels of AMH in follicles of varying stages between days 0 and 7 following GnRHa administration were quantified using immunohistochemistry. The expression of AMH in follicles at various stages revealed dynamic changes during the process of downregulation. AMH in primary follicles initially increased and then decreased gradually. In small and large preantral follicles and in granulosa cells (GCs) surrounding the oocyte of small antral follicles, the expression of AMH began to increase on day 1, was attenuated on day 2, and then increased to a peak. The expression levels of AMH in the GCs surrounding the basement membrane, in contrast to the GCs surrounding the oocyte, were significantly lower and did not increase on day 1. In all stages of follicles, the expression of AMH declined gradually between the peak level and last day of downregulation. On day 7, the varying follicular stages all expressed lower levels of AMH than on day 0. This decrease was more prominent in the higher dose groups, compared with the lower dose groups. In conclusion, GnRHa was observed to induce time­dependent changes in the expression of AMH at varying follicular stages, which occurred in a dose­dependent manner.

Zygotic G2/M cell cycle arrest induced by ATM/Chk1 activation and DNA repair in mouse embryos fertilized with hydrogen peroxide-treated epididymal mouse sperm.

Human sperm cryopreservation for assisted reproduction is compromised by ROS-induced sperm cryodamage. Our previous model study in which mouse sperm were treated with H2O2 to simulate sperm DNA-damage caused by cryopreservation-induced ROS have discovered that mouse embryos fertilized with treated sperm showed a delay in cleavage that might be associated with cell cycle arrest. The DNA-damage checkpoint pathway underlying the delay remained elusive. Moreover, our previous study have also indicated that γH2AX, the DNA-damage repair marker, was functional in mouse embryos similarly fertilized, but the completeness and correctness are unknown and warrant more studies because insufficiency of completeness and correctness of DNA repair would otherwise trigger apoptosis. Based on the aforementioned model, we used embryo culture, inverted microscope, BrdU incorporation and immunofluorescence to explore the cell cycle phase that arrest occurred and the underlying DNA-damage checkpoint pathway in mouse zygotes fertilized with H2O2-treated sperm. We also adopted Tunel to investigate the apoptosis of mouse embryos similarly fertilized at different developmental stages to testify the completeness and correctness of sperm-derived DNA-damage repair. We found G2/M cell cycle arrest in zygotes fertilized with H2O2-treated sperm. ATM (pSer-1981) and Chk1 (pSer-345) activations, rather than ATR (pSer-428) and Chk2 (pThr-68), were detected in zygotes of the treated group. The apoptosis of embryos of different developmental stages of the treated group weren't different from those of the untreated group. In conclusions, ATM (pSer-1981)-Chk1 (pSer-345) cascade might have mediated G2/M cell cycle arrest and allowed time to facilitate sperm-derived DNA-damage repair in mouse zygotes fertilized with oxygen-stressed sperm, and the DNA-damage repair might be effective.

Effects of the insemination of hydrogen peroxide-treated epididymal mouse spermatozoa on γH2AX repair and embryo development.

BACKGROUND: Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excessive reactive oxygen species (ROS) generation. METHODS AND FINDINGS: We used exogenous ROS (H(2)O(2)) to simulate cryopreservation and examined DNA damage repair in embryos fertilized with sperm with H(2)O(2)-induced DNA damage. Sperm samples were collected from epididymis of adult male KM mice and treated with capacitation medium (containing 0, 0.1, 0.5 and 1 mM H(2)O(2)) or cryopreservation. The model of DNA-damaged sperm was based on sperm motility, viability and the expression of γH2AX, the DNA damage-repair marker. We examined fertility rate, development, cell cleavage, and γH2AX level in embryos fertilized with DNA-damaged sperm. Cryopreservation and 1-mM H(2)O(2) treatment produced similar DNA damage. Most of the one- and two-cell embryos fertilized with DNA-damaged sperm showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed γH2AX in the one- and four-cell embryos. CONCLUSIONS: γH2AX may be involved in repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa.

Endocrine traits of polycystic ovary syndrome in prenatally androgenized female Sprague-Dawley rats.

Although hyperandrogenism is an important condition and is considered the possible pathogenesis behind polycystic ovary syndrome (PCOS), data supporting this is still scarce. We sought to determine whether or not prenatal androgen exposure leads to PCOS and the possible cellular mechanisms involved. To induce prenatal androgen exposure, pregnant rats were treated with daily subcutaneous injections of free testosterone (T) or dihydrotestosterone (DHT) from embryonic days 16 to 19, and their female offspring were studied as adults. The mRNA expression of the progesterone receptor (PR) in the preoptic area (POA) hypothalamus was higher in the experimental groups than in the control group after ovariectomy and stimulation with estradiol benzoate. The levels of T, P, leutinizing hormone (LH), and estradiol were higher in the experimental groups than in the control groups. The frequency and magnitude of LH secretion was increased in experimental rats as compared with the control group. The anogenital distance of the experimental groups was prolonged and the nipple number was lower than that of the control group. Almost all experimental rats had prolonged or irregular estrous cycles. The experimental groups had fewer corpus luteum and preovulatory follicles and more preantral follicles and antral follicles than the controls. Our findings are consistent with the hypothesis that excess androgen during the prenatal period may cause PCOS. Additionally, we show that hyperandrogenic interference in the release of preovulatory LH surges is mediated by the suppressive effects of androgens on PR expression in POA-hypothalamic tissue.

Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation.

Human semen cryopreservation in the clinical management of male infertility is complicated by cryodamage to spermatozoa. We aimed to clarify the full pattern of cryodamage and evaluate the protective effects of ascorbate and catalase on cryopreserved spermatozoa. Semen samples were collected from 30 fertile males. Each sample was divided into 6 groups: fresh semen, cryopreserved semen without treatment, and samples cryopreserved with ascorbate (300 or 600 µM) or catalase (200 or 400 IU/mL). Spermatozoa were examined for their viability, motility, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), apoptosis (positive for annexin V and negative for propidium iodide [ie, Ann(+)/PI(-)]), and DNA damage (Olive tail moment [OTM]) in the presence or absence of ascorbate or catalase during cryopreservation. In comparison with the fresh spermatozoa, there was a significant decrease in the viability, motility, and MMP but increase in Ann(+)/PI(-) and OTM in the cryopreserved spermatozoa (P < .01 and P < .05, respectively). Concurrently, ROS levels in the postthaw spermatozoa also increased significantly, and this elevation was well correlated with the quality variations of postthaw spermatozoa (P < .01 for all). Ascorbate (300 µM) and catalase (200 and 400 IU/mL) reduced the ROS levels in postthaw spermatozoa significantly, compared with those in the control (P < .05). Furthermore, these antioxidants also prevented those characteristics from being adversely affected (P < .05). This study demonstrated that cryopreservation results in cryodamage to human spermatozoa, possibly through the mechanism of ROS. Appropriate ascorbate or catalase supplementation of cryoprotective medium restrains ROS levels and the resultant cryodamage.

[Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure].

OBJECTIVE: To evaluate the protective effects of oxidative DNA damage by adding antioxidants: ascorbate, catalase (CAT), and superoxide dismutase (SOD) in human semen samples undergoing cryopreservation procedure. METHODS: Semen sample form 30 fertile men were mixed with modified cryoprotectant and divided into six groups according to the category and concentration of antioxidants: ascorbate 300 micromol/L, ascorbate 600 micromol/L, CAT 200 U/ml, CAT 400 U/ml, SOD 200 U/ml, and SOD 400 U/ml. Comet assay was conducted to measure the percentage of comet cells, and the nuclear DNA damaged parameters: tail DNA percentage (TD%) and Olive tail moment (OTM). Flow cytometry was used to detect the reactive oxidative species (ROS). The motility (a + b grade), viable recovery rate, nuclear DNA integrity and reactive oxidative species (ROS) of all groups were analyzed before and/or after freeze-thawing. RESULTS: (After cryopreservation, compared with the control group, the a + b grade sperm rates of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were all higher than that of the control group (all P < 0.05), however, the levels of reactive oxygen species (ROS) of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 30 +/- 13, 30 +/- 11, and 30 +/- 11 respectively, all significantly lower than that of the control group (37 +/- 17 , all P < 0.05). The viable recovery rates of the ascorbate 300 micromol/L , CAT 200 U, and CAT 400 U groups were 67% +/- 14%, 68% +/- 14%, and 69% -/+ 15% respectively, all significantly higher than that of the control group (59% +/- 10%, all P < 0.05). (2) The TD% levels of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 41% +/- 4%, 40% +/- 7%, 40% +/- 6%, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (46% +/- 6%, all P < 0.01). The OTM levels of the ascorbate 300 micromol/ L, CAT 200 U, and CAT 400 U groups were 7.7 +/- 1.2, 7.5 +/- 1.6, and 7.8 +/- 1.9, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (10.1 +/- 3.1, all P < 0.01) too. The TD% and OTM levels of the other groups were all significantly higher than that of the raw semen (all P < 0.01), but not significantly different from those of the control group (all P > 0.05). (3) ROS was significantly negatively correlated with the motility in all groups (P < 0.05 or P < 0.01). Apart from the ascorbate 600 micromol/L group, the TD% and OTM of the other groups were all significantly positively correlated with the ROS (P < 0.05 or P < 0.01). CONCLUSION: Supplementation of ascorbate or CAT reduces the level of ROS that induces sperm nuclear DNA damage, and improves the human sperm quality in the process of freeze-thawing.

Fertilization of IVF/ICSI using sibling oocytes from couples with subfertile male or unexplained infertility.

The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluated. A total of 410 sibling oocyte cumulus-corona complexes (OCCC) from 21 couples with subfertile male (group A) and 11 unexplained infertile couples (group B) were randomly divided, in order of retrieval, into two groups inseminated either by conventional IVF or by ICSI. The treatment outcomes and the influence of infertility factors on fertilization in each group were compared. The results showed that although the two pronuclear (2PN) fertilization rate per injected sibling oocytes was significantly higher after ICSI (group A: 68.2% +/- 28.8%; group B: 66.2% +/- 24.9%) than after conventional IVF (group A: 41.8% +/- 32.7%; group B: 40.1% +/- 22.1%), the other variables studied included: the fertilization rates of per allocated sibling oocytes IVF/ICSI, the fertilization rates of sibling oocytes IVF/ICSI after excluding failed IVF fertilization cycles, as well as the cleavage rates of normal fertilization were not statistically significant (P>0.05). Similarly, though the total fertilization failure rate in the IVF group (group A: 42.9%; group B: 36.4%) was significantly higher than in the ICSI group (group A: 4.8%; group B: 0), we did not cancel cycles due to the normal fertilization of sibling oocytes. Embryo transfer was possible in all 32 couples. There were 10 clinical pregnancies in the two groups. We also discovered a possible association between some semen parameters and sperm functions of group A, and women age and duration of infertility of group B and fertilization. It is suggested that adoption of the split IVF/ICSI technology in the above cases may help eliminate fertilization failures. This is also a useful method to investigate the effect of single factor on the employment of assisted reproductive technology.

Intracytoplasmic sperm injection in cases with history of in vitro fertilization failure.

AIM: To evaluate the effect of intracytoplasmic sperm injection (ICSI) in the management of cases with a history of conventional in vitro fertilization (IVF) failure. METHODS: Two groups of patients, 19 with normal semen parameters and a history of IVF failure (metaphase II oocytes: 0-30 %) and 28 with severe male factor infertility received ICSI technology during the same period. Ovarian stimulation was achieved by conventional procedure. Transvaginal ultrasound-guided oocyte collection was done 35-37 h after human chorionic gonadotrophin (hCG) injection. Only metaphase II oocytes were selected for microinjection. RESULTS: Fertilization was achieved with ICSI in all the patients. The fertilization rate (75.6 % +/-21.1 % vs. 73.9 % +/-19.2 %), cleavage rate (85.1 % +/-19.3 % vs. 82.7 % +/-22.1 %), clinical pregnancy rate per embryo transfer cycle (31.6 % vs. 28.6 %) and implantation rate per embryo (15.3 % vs. 14.4 %) did not differ significantly between the two groups. CONCLUSION: ICSI is a valuable method for couples with a history of IVF failure. These patients may have a similar ICSI result as in severe male infertility.