Superovulation procedures and assisted reproductive technologies have been widely used to treat couples who have infertility problems. Although generally safe, the superovulation procedures are associated with a series of complications, such as ovarian hyper-stimulation syndrome, thromboembolism, and adnexal torsion. The role of long-term repeated superovulation in ovarian aging and especially in associated disorders such as osteoporosis and cardiovascular diseases is still unclear. In this study, we sought to determine if repeated superovulation by ten cycles of treatment with pregnant mare serum gonadotropin/human chorionic gonadotropin could affect ovarian reserve, ovarian function, bone density and heart function. Ovarian reserve and function were reflected by the size of the primordial follicle pool, anti-Mullerian hormone expressions, hormone levels and fertility status. Furthermore, we examined bone density and heart function by microCT and cardiovascular ultrasonography, respectively. After repeated superovulation, the size of the primordial follicle pool and the expression of anti-mullerian hormone decreased, along with the concentrations of estrogen and progesterone. Mice exposed to repeated superovulation showed an obvious decrease in fertility and fecundity. Furthermore, both bone density and heart ejection fraction significantly decreased. These results suggest that repeated superovulation may increase the risk of osteoporosis and cardiovascular diseases by accelerating ovarian aging.

The effect of extending the length of the FSH treatment protocol on superovulatory response and embryo production was investigated in wood bison during the anovulatory and ovulatory seasons. In Experiment 1 (anovulatory season), follicular wave emergence was synchronized by follicular ablation (Day -1) and bison were assigned randomly to two groups (n = 14/group) and given 200 mg FSH on Day 0 and Day 2 (non-extended group), or 133 mg FSH on Days 0, 2, and 4 (extended group). Human chorionic gonadotropin (hCG; 3000 IU) was given on Day 5 and Day 6 in the non-extended and extended groups, respectively, and bison were inseminated 12 and 24 h later. Ova/embryos were collected 8 days after hCG treatment. In Experiment 2 (ovulatory season), bison were synchronized and superstimulated as in Experiment 1 (n = 12/group), but prostaglandin was given to control CL development. Data were compared by t-test and Chi-square test. In Experiment 1, no differences in ovarian response or embryo production between groups were detected. In Experiment 2, there was no difference in the ovarian response between groups, however, a greater number of ova/embryos (4.3 ± 0.8 vs. 2.3 ± 0.4; P ≤ 0.05) and freezable embryos (2.5 ± 0.6 vs. 1.2 ± 0.4; P ≤ 0.05) were obtained in the extended group. The number of freezable embryos was greater during the ovulatory vs anovulatory season (1.8 ± 0.4 vs. 0.3 ± 0.2; P ≤ 0.05). In conclusion, extending the FSH treatment in wood bison did not improve the superovulatory response during the anovulatory season, but resulted in twice as many freezable embryos during the ovulatory season. The number of freezable embryos collected during the anovulatory season was <20% that of the ovulatory season.

Chronic treatment with gonadotrophin-releasing hormone (GnRH) agonist eliminates luteinizing hormone (LH) pulses and inhibits maturation of Graafian follicles in sheep. Since the presence of 'dominant' follicles may inhibit superovulatory responses, an experiment was conducted to determine whether a GnRH agonist could be used in conjunction with follicle-stimulating hormone (FSH) to induce a superovulatory response with production of normal embryos. Twenty-four Welsh Mountain ewes were chronically treated with GnRH agonist by means of a subcutaneous minipump. Twelve of the ewes were given 12 mg progesterone intramuscularly (i.m.) twice daily for four days; all ewes were then given 672 micrograms (total) of highly purified FSH continually infused intravenously for either 72 h (Group A) or 96 h (Group B) in a 2 x 2 experimental design (n = 6). Ovulation was then induced with 750 I.U. human chorionic gonadotrophin injected i.m. (Day 0) and all ewes were inseminated into the uterus with > 100 x 10(6) fresh sperm on Day 0. Embryos were flushed from the uterus, and ovaries were inspected at laparotomy on Day 5. Pretreatment with progesterone did not affect any of the parameters measured and data were pooled accordingly. There were no differences between Groups A and B in the number of ovulations or the number of embryos recovered, although there were more large unruptured follicles in Group A animals (8.8 +/- 0.8 v. 3.1 +/- 0.7, P < 0.001). The embryo recovery rate was higher in Group A ewes (52.5 v. 26.4, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

High FSH doses during superovulation of heifers with a small ovarian reserve increase the number of dysfunctional ovulatory-size follicles that do not ovulate in response to human chorionic gonadotropin (hCG). Thus, anti-Müllerian hormone (AMH) and antral follicle count (AFC), two well-established biomarkers of responsiveness of individuals to superovulation, are hypothesized to be positively linked to number of dysfunctional ovulatory-size follicles developing in response to superovulation with high FSH doses. To test this hypothesis, heifers with a small ovarian reserve were stimulated beginning on Day 1 of the estrous cycle with twice daily treatments for 4 days with each of four Folltropin-V (FSH) doses (35 IU, 70 IU (industry standard), 140 IU, or 210 IU) followed by prostaglandin F2α to regress corpora lutea (CL) from the previous estrous cycle and hCG to induce ovulation. Ovulatory-size follicles were classified as functional or dysfunctional based on whether they ovulated and formed CL in response to hCG. FSH dose did not impact the relationship between AMH, AFC and the number of functional or dysfunctional ovulatory-size follicles developing in response to superovulation. Thus, data from the four superovulations were averaged for each heifer. AMH and AFC were positively associated with the subsequent number of functional and dysfunctional ovulatory-size follicles and the proportion of ovulatory-size follicles that are dysfunctional after superovulation. Because measurements of AMH concentration and AFC predict the number but not functionality of ovulatory-size follicles, which may also impact oocyte quality, these ovarian reserve biomarkers are concluded to be unlikely useful to improve IVF or embryo transfer outcomes in heifers with a small ovarian reserve.

Juvenile superovulation can provide a wealth of oocyte material for embryo production, animal cloning, and genetic modification research, but embryos derived from juvenile oocytes show poor efficiency in subsequent developmental capacity. In order to reveal the formation mechanism of large numbers of follicles and poor oocyte quality in juvenile ovaries under superovulation treatment, differentially expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) were characterized and investigated in the ovaries of lambs and adult sheep using high-throughput sequencing technology. The majority of differentially expressed miRNAs (337/358) were upregulated in lamb libraries. The expression levels of mRNAs related to hormone receptors (follicle-stimulating hormone receptor, FSHR; luteinizing hormone/choriogonadotropin receptor, LHCGR; estrogen receptor 1, ESR1), steroid hormone secretion (cytochrome P450 family 11 subfamily A member 1, CYP11A1; cytochrome P450 family 17 subfamily A member 1, CYP17A1; cytochrome P450 family 19 subfamily A member 1, CYP19A1), and oocyte quality (pentraxin 3, PTX3; BCL2 apoptosis regulator, BCL2; caspase 3, CASP3) were significantly different between the lamb and adult libraries. The miRNA aor-miR-143, which targets FSHR, was highly and differentially expressed, and PTX3 was predicted to be targeted by oar-miR-485-3p and oar-miR-377-3p in the ovine ovary. A considerable number of miRNAs were predicted to inhibit ESR1 expression in lamb ovaries. In conclusion, oar-miR-143 and FSHR molecules, among others, might regulate follicle formation, and oar-miR-485-3p, oar-miR-377-3p, and PTX3, among others, may be associated with oocyte quality. These identified miRNAs and mRNAs will be beneficial for the prediction of ovarian superovulation potential and screening of oocytes.

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.

The aim of this randomized controlled trial was to compare the efficacy of extended letrozole regimen with clomiphene citrate in women with unexplained infertility undergoing superovulation and intrauterine insemination (IUI).

To evaluate the effects of He's Yangchao Recipe (HSYC) on ameliorating ovarian oxidative stress of aging mice under consecutive superovulation.

In vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART).

Mature oocytes at the metaphase II status (MII-stage oocytes) played an important role in assisted reproductive technology in non-human primates.

The aim of this study was to devise an efficient technique for generating embryos from high-quality bovine females. Oocytes were collected from 20 control and 15 Hanwoo (Bos taurus coreanae) females treated with the FSH. A combination of decreasing FSH doses (36, 36, 24, and 24 mg, 12 h apart), progesterone, estrogen, and prostaglandins were administered to synchronize and mildly stimulate the animals. The FSH-treated group (1125 oocytes) and control group (1022 oocytes) exhibited a higher proportion of grade A and B oocytes (88.2%) than the other grades (p < 0.05), with most at the germinal vesicle 2 stage (64.0%). Moreover, the FSH-treated group achieved a notably higher blastocyst rate (44.7%) compared to the control group (31.1%) (p < 0.05). After undergoing vitrification and in vitro culture (IVC) warming, embryos in the FSH group exhibited higher re-expansion rates (grade 1: 86.9%; grades 2 and 3: 57.9%) compared to those in the control (p < 0.05). This highlights the positive impact of FSH treatment on in vitro embryo production (IVEP) and the OPU rate.

Technologies that can preselect offspring gender hold great promise for improving farm animal productivity and preventing human sex-related hereditary diseases. The maternal Rlim allele is required for imprinted X-chromosome inactivation, which is essential for the normal development of female mouse embryos. In this study, we inactivated the maternal Rlim allele in embryos by crossing a male transgenic mouse line carrying an X-linked CMV-Cre transgene with a female line carrying a loxP-flanked Rlim gene. Knockout of the maternal Rlim gene in embryos resulted in a male-biased sex ratio skew in the offspring. However, it also reduced litter size, and this effect was not compensated for by superovulation in the mother mice. In addition, we showed that siRNA-mediated knockdown of Rlim in mouse embryos leads to the birth of male-only progenies. This study provides a new promising method for male-biased sex selection, which may help to improve the productivity in livestock and prevent sex-associated hereditary diseases in humans.

Metformin, an oral biguanide traditionally used for the treatment of type 2 diabetes, is widely used for the management of polycystic ovary syndrome (PCOS)-related anovulation. Because of the significant prevalence of insulin resistance and glucose intolerance in PCOS patients, and their putative role in ovulatory dysfunction, the use of metformin was touted as a means to improve ovulatory function and reproductive outcomes in PCOS patients. To date, there has been inconsistent evidence to demonstrate a favorable effect of metformin on oocyte quality and competence in women with PCOS. Given the heterogeneous nature of this disorder, we hypothesized that metformin may be beneficial in mice with aberrant metabolic characteristics similar to a significant number of PCOS patients. The aim of this study was to gain insight into the in vitro and in vivo effects of metformin on oocyte development and ovulatory function.

The conventional method of ovarian superstimulation requires multiple injections of gonadotropins which is time-consuming and may be stressful for the cows. This study was designed to determine whether a single epidural injection of FSH (EI group) would induce the superovulatory response in the Thai-Holstein crossbreed and evaluate FSH plasma hormone concentrations. Eight cows (replication = 3; n=24) were assigned to one of 2 treatments in switch back design. Control group (n=12): cows were received 400 mg FSH twice daily by intramuscularly for 4 days (80, 80, 60, 60, 40, 40, 20 and 20 mg), EI group (n=12): cows were received 400 mg FSH by single epidural injection. Data were collected in term of ovarian follicle responses, superovulatory responses, ova/embryo collection. FSH concentrations were examined using ELISA. The total follicular responses during oestrus were not different between treatments; however, the large follicles were less frequent (P < 0.01) while the medium follicle sizes were higher (P < 0.05) in the EI group. The plasma concentration of FSH in EI was dramatically increased within 2 hours before decreasing sharply thereafter (P < 0.01) and did not remain above baseline after 10 hours of administration. The embryo quality was better in the control than the EI groups (P < 0.05). Interestingly, the number of ovulation cysts in the EI group was 50%. The ovarian responses and embryo quality in the cows with cysts were worse compared with the non-cyst groups (P < 0.05). In conclusion, alternative protocols decreased the superovulatory response and increased poor embryo quality in Thai-Holstein crossbred. Also, the incidence of ovarian follicular cysts is higher in the EI group.

In their attempt to fulfill the wish of having children, women who suffer from fertility issues often undergo assisted reproductive technologies such as ovarian stimulation, which has been associated with adverse health outcomes and imprinting disorders in children. However, given the crucial role of exogenous hormone stimulation in improving human infertility treatments, a more comprehensive analysis of the potential impacts on DNA methylation in embryos following ovarian stimulation is needed. Here, we provide genome-wide DNA methylation profiles of blastocysts generated after superovulation of prepubertal or adult mice, compared with blastocysts derived from non-stimulated adult mice. Additionally, we assessed the impact of the in vitro growth and maturation of oocytes on methylation in blastocysts.

Assisted reproductive technologies are fertility treatments used by subfertile couples to conceive their biological child. Although generally considered safe, these pregnancies have been linked to genomic imprinting disorders, including Beckwith-Wiedemann and Silver-Russell Syndromes. Silver-Russell Syndrome is a growth disorder characterized by pre- and post-natal growth retardation. The Mest imprinted domain is one candidate region on chromosome 7 implicated in Silver-Russell Syndrome. We have previously shown that maintenance of imprinted methylation was disrupted by superovulation or embryo culture during pre-implantation mouse development. For superovulation, this disruption did not originate in oogenesis as a methylation acquisition defect. However, in comparison to other genes, Mest exhibits late methylation acquisition kinetics, possibly making Mest more vulnerable to perturbation by environmental insult. In this study, we present a comprehensive evaluation of the effects of superovulation and in vitro culture on genomic imprinting at the Mest gene. Superovulation resulted in disruption of imprinted methylation at the maternal Mest allele in blastocysts with an equal frequency of embryos having methylation errors following low or high hormone treatment. This disruption was not due to a failure of imprinted methylation acquisition at Mest in oocytes. For cultured embryos, both the Fast and Slow culture groups experienced a significant loss of maternal Mest methylation compared to in vivo-derived controls. This loss of methylation was independent of development rates in culture. These results indicate that Mest is more susceptible to imprinted methylation maintenance errors compared to other imprinted genes.

This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compared with PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between AIS-hCG and PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females was decreased compared with PMSG-hCG (P<0.001). The PVS of oocytes from the AIS-hCG was larger than with PMSG-hCG (P<0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal, although there was an increased fluorescence intensity in the AIS-hCG oocytes (P<0.001). The F-actin and CGs distribution in oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). Although the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG oocytes showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggested that AIS-hCG superovulation produces high numbers of morphologically normal oocytes that also possess normal subcellular structures, good morphological characteristics and had high invitro embryonic developmental potential.

With the medical advancement some studies put forward that letrozole (LE), a specific aromatase inhibitor with the function of reducing oestrogen synthesis, has recently been applied as a potentially better alternative compared with clomiphene citrate (CC), owing to that it has a superior efficacy as compared with CC in patients of unexplained infertility undergoing intrauterine insemination (IUI). However, there is no one study can clear and definite whether LE can replace the CC as first line drug.

Superovulation is a reproductive technique generally used to produce genetically engineered mice. Superovulation in mice involves the administration of equine chorionic gonadotropin (eCG) to promote follicle growth and then that of human chorionic gonadotropin (hCG) to induce ovulation. Previously, some published studies reported that inhibin antiserum (IAS) increased the number of ovulated oocytes in ddY and wild-derived strains of mice. However, the effect of IAS on the C57BL/6 strain, which is the most widely used inbred strain for the production of genetically engineered mice, has not been investigated. In addition, the combined effect of IAS and eCG (IASe) on the number of ovulated oocytes in superovulation treatment has not been examined. In this study, we examined the effect of IAS and eCG on the number of ovulated oocytes in immature female mice of the C57BL/6 strain in superovulation treatment. Furthermore, we evaluated the quality of obtained oocytes produced by superovulation using IASe by in vitro fertilization (IVF) with sperm from C57BL/6 or genetically engineered mice. The developmental ability of fresh or cryopreserved embryos was examined by embryo transfer. The administration of IAS or eCG had a similar effect on the number of ovulated oocytes in C57BL/6 female mice. The number of ovulated oocytes increased to about 3-fold by the administration of IASe than by the administration of IAS or eCG alone. Oocytes derived from superovulation using IASe normally developed into 2-cell embryos by IVF using sperm from C57BL/6 mice. Fresh or cryopreserved 2-cell embryos produced by IVF between oocytes of C57BL/6 mice and sperm from genetically engineered mice normally developed into live pups following embryo transfer. In summary, a novel technique of superovulation using IASe is extremely useful for producing a great number of oocytes and offspring from genetically engineered mice.

The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n = 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SC group; n = 6) or sows with postweaning spontaneous estrus (NC group; n = 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes ≥2 or ≤2, P value <0.05). Hormonal treatments almost doubled (P < 0.03) the number of corpora lutea (39.8 ± 10.2 and 38.3 ± 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 ± 3.8). In contrast, embryo viability significantly decreased (P < 0.003) in response to SS-7 treatment (75.1% ± 15.2%) compared to SC and NC groups (93.8 ± 7.6% and 91.8 ± 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/β-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj, and Usp8). A key finding in this study was the downregulation of β-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo-uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo-maternal crosstalk.