Relationship between superovulation and embryo production with ovarian follicular population before superovulatory treatment in Brazilian Bergamasca sheep / [Relação da resposta superovulatória e produção embrionária com a população folicular ovariana antes do tratamento superovulatório de ovelhas da raça Bergamácia Brasileira]

The objective of this study was to quantify the superovulatory response and embryo production of Brazilian Bergamasca sheep and to evaluate the link to the follicular condition before superovulatory treatment, as a reference for selection of donors with potential for superovulation. Follicular population of twenty-three sheep was evaluated by ultrasound during metestrus phase of the estrous cycle and divided into groups of low, medium and high follicular population. Subsequently, they were synchronized, superovulated with 133mg of pFSH, mated and subjected to embryo collection. The superovulatory response (9.0±3.3 vs 10.7±6.2 vs 13.8±7.1) and embryo production (4.0±3.8 vs 2.6±2.0 vs 1,8±4.0) were similar between groups (P>0.05). There was a positive correlation between the number of follicles during the metestrus phase and the number of corpus luteum with premature regression (PLR) (0.52) and a negative correlation between the recovery rate and PLR (-0.44) (P<0.05). The sheep that presented PLR had more follicles during metestrus (16.9±7.8 vs 12.7±3.2) and lower embryo recovery rate (38.8±29.3 vs 72.2±29.9) than those with functional CLs (P<0.05). Follicular quantification during metestrus phases was unable to identify donors with high embryo production. Animals with PLR had a larger follicular population during metestrus and lower embryo recovery rate.(AU)

O objetivo deste trabalho foi quantificar a resposta superovulatória e a produção embrionária de ovelhas Bergamácia Brasileira e relacioná-las com a condição folicular antes do tratamento superovulatório, como referência para seleção de doadoras com potencial para superovulação. Vinte e três ovelhas foram avaliadas quanto à população folicular por ultrassonografia na fase de metaestro do ciclo estral e divididas em grupos com baixa, média e alta população folicular. Posteriormente foram sincronizadas, superovuladas com 133mg de pFSH, acasaladas e submetidas à coleta de embriões. A resposta superovulatória (9,0±3,3 vs. 10,7±6,2 vs. 13,8±7,1) e a produção embrionária (4,0±3,8 vs. 2,6±2,0 vs. 1,8±4,0) foram semelhantes entre os grupos (P>0,05). Houve correlação positiva entre o número de folículos no metaestro e o número de corpos lúteos com regressão prematura (RPCL) (0,52) e correlação negativa entre a taxa de recuperação e RPCL (-0,44) (P <0,05). As ovelhas que apresentaram RPCL tiveram mais folículos no metaestro (16,9±7,8 vs. 12,7±3,2) e menor taxa de recuperação embrionária (38,8±29,3 vs. 72,2±29,9) do que as que apresentaram CLs funcionais (P<0,05). A quantificação folicular nas fases de metaestro não foi capaz de identificar doadoras com alto potencial de produção embrionária. Animais com RPCL tiveram maior população folicular no metaestro e menor recuperação de embriões.(AU)

Impact of Kuntai Capsules on LIF, IGF-1 and EGF expression in the implantation window of endometrium in mice / Impacto de cápsulas de Kuntai en la expresión de LIF, IGF-1 y EGF en la ventana de implantación de endometrio en ratones

To investigate the influence of Kuntai capsules on the expression level of leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-1)and epidermal growth factor (EGF) during the mouse's implantation window of superovulation period and controlled ovarian hyperstimulation period. 90 female mice were randomly divided into six groups in control, superovulation and controlled ovarian hyperstimulation (COH) conditions. The RNA expression of EGF, LIF and IGF-1 in the endometrium on the 4th day of pregnancy was detected, and the relative expression was compared. mRNA expression of these three factors in endometrium was significantly lower in superovulation and COH groups than control group (p<0.001). mRNA expression of these three factors in endometrium remained obviously lower in superovulation plus kuntai capsule group and COH plus kuntai capsule group than control group (p<0.01). mRNA expression of these three factors in endometrium was lower in control group than in the NS plus kuntai capsule group (p<0.05). Kuntai capsule cannot completely reverse the endometrial damages caused by superovulation and COH. Thus Kuntai capsule could partially improve a mouse's endometrial receptivity during the implantation window.

Para investigar la influencia de las cápsulas de Kuntai en el nivel de expresión del factor inhibidor de la leucemia (LIF), el factor de crecimiento similar a la insulina I (IGF-1) y el factor de crecimiento epidérmico (EGF) durante la ventana de implantación del ratón del período de superovulación y la hiperestimulación ovárica controlada período, se dividieron aleatoriamente 90 ratones hembra en seis grupos en condiciones de control, superovulación e hiperestimulación ovárica controlada (COH). Se detectó la expresión de ARN de EGF, LIF e IGF-1en el endometrio al cuarto día de embarazo, y se comparó la expresión relativa. La expresión de ARNm de estos tres factores en el endometrio fue significativamente menor en los grupos de superovulación y COH que en el grupo control (p<0,001). La expresión de ARNm de estos tres factores en el endometrio permaneció más baja en el grupo de cápsulas de superovulación más Kuntai y en el grupo de cápsulas de COH más Kuntai respecto del grupo control (p<0,01). La expresión de ARNm de estos tres factores en el endometrio fue menor en el grupo control que en el grupo de cápsula NS más Kuntai (p<0,05). La cápsula de Kuntai no pudo revertir completamente los daños endometriales causados por la superovulación y la COH. Por lo tanto, se sugiere que la cápsula de Kuntai podría mejorar parcialmente la receptividad endometrial de un ratón durante la ventana de implantación.

Respuesta superovulatoria en vacas donantes Brahman usando ablación folicular previo a protocolos de superovulación / Superovulatory response in Brahman donor cows using follicular ablation before superovulation protocols

RESUMEN Objetivo. Determinar el efecto de la ablación folicular en el inicio de un protocolo de superovulación (SPO) sobre la respuesta superovulatoria en vacas donantes de raza Brahman. Materiales y métodos. Se utilizaron 20 vacas de raza Brahman, las cuales fueron distribuídas aleatoriamente en dos grupos: Grupo control (G1; n = 10), la sincronización de la onda de crecimiento folicular fue realizada mediante la combinación de estrógenos (2.5 mg, Benzoato de Estradiol) y progestágenos (1 gr, implante intravaginal); cuatro días después se inició el protocolo de SPO con la hormona folículoestimulante porcina (FSHp); y grupo ablación (G2; n = 10), se realizó la ablación folicular y un día después se inició el tratamiento de SPO con FSHp . En los dos grupos la colecta de los embriones se realizó siete días después de la primera inseminación artificial. Resultados. El G2 presentó una mayor proporción de embriones de calidad 1 (p<0.01) en comparación con el G1 (68.60%, 31.22%), mientras que los animales del grupo G1 presentaron una mayor proporción de embriones de calidad 2 (43.04%, 18.60%, p<0.01). Para las variables total de estructuras colectadas, y total de embriones transferibles, no se observaron diferencias significativas (p>0.05). Conclusiones. La ablación folicular aumentó el porcentaje de embriones de calidad 1, sugiriendo que la implementación de esta técnica, como estrategia para sincronizar el inicio de una nueva onda de crecimiento folicular en tratamientos de SPO, mejora la calidad de los embriones producidos en vacas donadoras Brahman.

ABSTRACT Objective. The objective of this study was to determine the effect of follicular ablation at the beginning of a superovulation protocol (SOP) on the superovulatory response of Brahman donor cows. Materials and methods. Twenty Brahman cows were used, randomly distributed in two groups: control group (G1, n = 10), synchronization of the follicular growth wave was performed by the combination of estrogens (2.5 mg, estradiol benzoate) and progestagens (1 gr intravaginal implant); four days after starting the SOP with porcine follicle stimulating hormone (FSHp); and the ablation group (G2, n = 10), follicular ablation was performed and one day after, the SOP treatment with FSHp was initiated. In both groups, embryo collection was performed seven days after the first artificial insemination. Results. The G2 had a higher proportion of quality 1 embryos (p<0.01) compared to G1 (68.60% vs. 31.22%), while animals of G1 group had a higher proportion of quality 2 embryos (43.04% vs. 18.60%, p<0.01). For the total of structures collected and the total of transferable embryos, no significant differences were observed (p>0.05). Conclusions. Follicular ablation increased the percentage of quality 1 embryos, suggesting that the implementation of this technique, as a strategy to synchronize the beginning of a new wave of follicular growth when using SOP, improve embryo quality in Brahman donor cows.

Induction of superovulation in mature mice and rats using serum of spayed female dogs / 한국실험동물학회지

The following experiments were designed to examine the effect of serum of spayed dogs on superovulation response in mice and rats. In Experiment 1, female mice at diestrus (n=30) were divided into three equal groups and superovulated with either administration of 5 IU pregnant mare serum gonadotropin (PMSG) or recombinant follicle stimulating hormone (rFSH) (reducing dose from 2.5 to 0.5 IU) and 5 IU human chorionic gonadotropin (hCG) administered 48h later. Serum of spayed dogs was administered intraperitoneally at a reduced dose from 0.1 to 0.025 mL in a 48 h period. In Experiment 2, female rats (n=30) at diestrus stage were divided into three equal groups. Superovulation was induced using either 30 IU PMSG, or a dose reduced from 5 to 1 IU rFSH and 25 IU hCG administered 48h later. Serum of spayed dogs was administered in a reduced dose from 0.6 to 0.1 mL in a 48 hour period. Female mice and rats were mated 24 h following hCG administration. On day 14 after mating, animals were euthanized and ovarian sections were fixed for histopathological evaluation and corpus luteum (CL) counting. No significant difference observed in mean (±SEM) number of CLs between the PMSG group and the mice that received serum of spayed dog (10.4±1.3 vs 9.2±1.0). Mean (±SEM) number of CLs tended to be lower in rats that received serum of spayed dog than those of rats which received either PMSG or rFSH (15.1±1.9 vs 23.6±3.1 and 23.1±2.9, P=0.06, respectively). In conclusion, serum of spayed dogs is able to induce a superovulatory response in mice and rats.

The likelihood of achieving pregnancy through timed coitus in young infertile women with decreased ovarian reserve / 대한생식의학회지

OBJECTIVE: To evaluate the pregnancy rate and time to pregnancy after timed coitus with or without superovulation in infertile young women younger than 35 years old with low serum anti-Müllerian hormone (AMH) levels (< 25th percentile). METHODS: A total of 202 patients younger than 35 years old were recruited retrospectively between 2010 and 2012. Ninety-eight women had normal serum AMH levels (25–75th percentile), 75 women had low serum AMH levels (5th≤&< 25th percentile) and 29 women had very low serum AMH levels (< 5th percentile), according to reference values for their age group. RESULTS: The clinical pregnancy rate was positively associated with AMH levels, but this trend did not reach statistical significance (43.9% vs. 41.3% vs. 27.6% in the normal, low, and very low AMH groups, respectively). The time to pregnancy was longer in the very low AMH group than in the normal AMH group (13.1±10.9 months vs. 6.9±6.1 months, p=0.030). The cumulative live birth rate over 18 months was lower in the very low AMH group than in the normal AMH group, with marginal significance (20.0% vs. 55.9%, p=0.051). The duration of infertility was negatively correlated with achieving pregnancy (odds ratio, 0.953; 95% confidence interval, 0.914–0.994; p=0.026). CONCLUSION: Conservative management, such as timed coitus with or without superovulation, should be considered in young patients who have low ovarian reserve without any infertility factors. However, for women with a long duration of infertility or very low serum AMH levels, active infertility treatment should be considered.

Estudo da vascularização folicular e do corpo lúteo de éguas cíclicas tratadas com extrato de pituitária equina utilizando ultrassom Doppler colorido / Study of follicular and corpus luteum vascularization in mares treated with Equine Pituitary Extract using ultrasound color Doppler

Informações sobre a vascularização da parede folicular e do corpo lúteo equino, associadas à superovulação, são escassas. Com o objetivo de avaliar o efeito superovulatório do extrato de pituitária equina (EPE) no fluxo sanguíneo folicular e luteal, foram utilizadas seis éguas Puro Sangue Árabe, em dois ciclos estrais (controle e tratamento). As éguas foram monitoradas diariamente por ultrassonografia modo B, até que os folículos atingissem diâmetro de 23mm (desvio). No ciclo tratamento, as éguas receberam 8mg de EPE, uma vez ao dia, por via IM, até que dois ou mais folículos atingissem o diâmetro entre 32 e 35mm. A ovulação foi induzida com acetato de deslorelina, quando os folículos atingiram, no mínimo, 35mm. No momento do desvio folicular, da indução da ovulação e do último exame pré-ovulatório, foi utilizada a ultrassonografia modo B para medir o diâmetro dos folículos e, no oitavo dia pós-ovulação, para a área do corpo lúteo (CL). Utilizou-se também ultrassonografia com Doppler colorido para avaliar a perfusão sanguínea da parede folicular e do parênquima luteal. No ciclo controle, foi realizado o mesmo procedimento, exceto pelo uso do EPE. Os dados foram submetidos à análise de variância, com nível de significância de 5%. Não foi observado efeito do EPE sobre o número de ovulações, o diâmetro dos folículos, a vascularização da parede folicular e a concentração sérica de estrógeno. Os animais, tratados ou não, apresentaram CLs funcionais, não havendo diferença na área do parênquima ou da vascularização luteal, nem na concentração sérica de progesterona, no oitavo dia após a ovulação. Foi observado que o EPE proporcionou um maior número de folículos subordinados no momento da indução da ovulação do folículo dominante (P ≤ 0,05). Embora esses folículos não tenham chegado a ovular, concluiu-se que o EPE atuou no crescimento de folículos, que podem ser utilizados em outras biotécnicas, como a transferência de oócitos, com maior aproveitamento da reserva folicular de ovários equinos.(AU)

Knowledge about follicle and corpus luteum vascularization associated with superovulation in mares is scarce. Aiming to evaluate the effect of equine pituitary extract (EPE) on superovulation, the experiment was conducted using six mares Purebred Arabian in two estrous cycles (control and treatment). The mares were synchronized, and monitored daily by ultrasound B mode until the follicles reached diameter ≤ 23 mm (deviation). In the treatment cycle, from the deviation, mares received 8 mg of EPE, once a day, intramuscularly, until two or more follicles reached a diameter between 32 and 35 mm. Ovulation was induced with deslorelin acetate when follicles reached at least 35 mm. At the time of follicular deviation, induction of ovulation and final preovulatory exam, it was used B-mode ultrasound to measure the diameter of follicles and on the eighth day after ovulation to measure the area of the corpus luteum (CL); color Doppler was also used to assess blood perfusion of the follicle wall and luteal parenchyma. In the control cycle was performed the same procedure except for the use of EPE. Data were subjected to analysis of variance, with 5% significance level. There was no effect of EPE on ovulation number, diameter of follicles, vascularity of the follicular wall and serum estrogen concentration. The animals treated or not, showed functional CLs, with no difference in parenchymal area or luteal vascularization, or in serum progesterone concentration on the eighth day after ovulation. It was observed that the EPE provided a greater number of subordinate follicles at the time of induction of ovulation of the dominant follicle. Although these follicles have failed to ovulate, it was concluded that EPE influenced the follicles growth, and it can be used in other biotechnologies, with greater utilization of equine ovarian follicular reserve.(AU)

Repeated superovulation induction after failure in previous IVF-ET cycles with the ultra-long protocol: Analysis of outcomes of clinical pregnancy / 中华男科学杂志

Objective@#To analyze the clinical outcomes of repeated superovulation induction in patients with adenomyosis or moderate to severe pelvic endometriosis after failure in previous IVF-ET cycles with the ultra-long protocol.@*METHODS@#We retrospectively analyzed the clinical data about 37 patients with adenomyosis or moderate to severe pelvic endometriosis in our center from 2009 to 2013, who underwent repeated IVF-ET after failure in the previous cycles with the ultra-long protocol, namely by injection of 2－6 ampoules of 3.75 mg gonadotropin-releasing hormone agonist (GnRH-a). All the patients met the following requirements: hCG-negative at 14 days after transfer, within 3－7 days after menstruation, and properly down-regulated serum follicle stimulating hormone (FSH) (<10 mIU/ml), luteinizing hormone (LH) (<10 mIU/ml), estradiol (E2) (<30 pg/ml), follicle diameter (<10 mm) and endometrial thickness, and received GnRH (Gonal-F, Serono) for ovulation induction. We compared the clinical and laboratory data and pregnancy outcomes between the first and repeated cycles before and after ovulation induction.@*RESULTS@#The repeated cycles, as compared with previous ones, showed significant increases in the antral follicle count (AFC) on the first day of stimulation (7.55 ± 1.86 vs 6.45 ± 2.5, P<0.05), number of follicles =≥14 mm in diameter on the hCG trigger day (7.81 ± 3.6 vs 5.56 ± 3.68, P<0.05), level of E2 (［2 362.15 ± 1 210.49］ vs ［1 749.22 ± 1 139.44］ pg/ml, P<0.05), and numbers of oocytes retrieved (7.51 ± 3.23 vs 4.78 ± 3.41, P<0.05) and embryos transferred (2.00 ± 0.33 vs 1.50 ± 0.67, P<0.05), exhibited a remarkably reduction in the dose of GnRH (［1 791.65 ± 1 889.41］ vs ［3 439.56 ± 1 836.53］ IU, P<0.05), and achieved a clinical pregnancy rate of 62.16%.@*CONCLUSIONS@#With proper reduction of the FSH, LH and E2 levels and follicle diameter, repeated superovulation induction for IVF-ET can improve the ovarian response and pregnancy outcomes of the patients with adenomyosis or moderate to severe pelvic endometriosis after failure in the previous IVF-ET cycles with the ultra-long protocol.

Super pregnancy in a BALB/c mouse superovulated with PMSG / 한국실험동물학회지

This paper reports a case of super pregnancy in a BALB/c mouse pregnant with 30 pups following induction of superovulation using a PMSG-hCG protocol. Superovulation was induced in 10 mice by injecting 5 IU PMSG followed by 5 IU hCG 48 hours later. Immediately after injection of hCG, animals were placed with males at a ratio of 1 to 1 for 24 hours. On day 14 after mating, animals were killed by cervical dislocation and the uterus was examined for pregnancy and the number of fetuses. The mean (±SEM) number of fetuses observed in ten mice was 5.4±3.18 with an unexpectedly super pregnant mouse bearing 30 fetuses on day 14 of pregnancy.

Produção de embriões em ovinos Morada Nova e Somalis Brasileira / Embryo production in Morada Nova and Somalis Brasileira ewes

Superovulatory response and embryo yield in 19 Morada Nova and 20 Somalis Brasileira ewes was analyzed. All animals were synchronized with the insertion of an intravaginal device (CIDR®) on Day 0, replaced by a new device on Day 7, which remained in place until Day 14 and superovulated with 133mg of porcine FSH (pFSH) in decreasing doses at 12h intervals from Day 12 until Day 15 of the treatment, and a single dose of equine chorionic gonadotropin (eCG, 200UI) on Day 14 (i.e., administered in CIDR removal). Fifty hours after CIDR® removal, females were inseminated by laparoscopy. All embryos were recovered by laparotomy 5 days after insemination. Sheep which responded to the superovulation protocol (P>0.05) included 74% of the Morada Nova ewes and 50% of the Somalis Brasileira ewes. Morada Nova showed better results (P<0.05) than Somalis Brasileira in number of ovulations (15.38 ± 5.24 vs. 10.56 ± 2.83), total structures (11.00 ± 7.55 vs. 3.33 ± 1.94) and embryo yields (6.79 ± 5.35 vs. 2.90 ± 2.18). Despite the high fertilization rate, degenerate embryo rate was high for both breeds, with an overall rate of 39% (57/145). In conclusion, superovulatory response and embryo yields in Morada Nova ewes were considered sufficient to justify the use of this procedure in genetic resources conservation programs. However, improvements to embryo quality and control of precocious regression of corpus luteum are necessary to produce better results in the MOET program, with minimal variations and maximum embryo yield in Morada Nova and Somalis Brasileira ewes.(AU)

Pretreatment of normal responders in fresh in vitro fertilization cycles: A comparison of transdermal estradiol and oral contraceptive pills / 대한생식의학회지

OBJECTIVE: The aim of this study was to investigate the impact of pretreatment with transdermal estradiol (E₂) compared to oral contraceptive pills (OCPs) on controlled ovarian stimulation (COS) response in normal responders undergoing fresh in vitro fertilization (IVF)-embryo transfer (ET) cycles. METHODS: A retrospective cohort study was performed of normal responders undergoing fresh IVF-ET cycles who received pretreatment with transdermal E₂ versus OCPs prior to fresh IVF-ET. The total days of ovarian stimulation, total dosage of gonadotropins, total number of oocytes, and mature oocytes retrieved were noted. Pregnancy outcomes after ET were also recorded. RESULTS: A total of 2,092 patients met the inclusion criteria: 1,057 and 1,035 patients in the transdermal E₂ and OCP groups, respectively. Patients in the OCP group had a longer duration of COS (10.7±1.63 days, p<0.01) than the E₂ group (9.92±1.94 days). Patients in the OCP group also required higher cumulative doses of gonadotropins (2,657.3±1,187.9 IU) than those in the E₂ group (2,550.1±1,270.2 IU, p=0.002). No statistically significant differences were found in the total and mature oocytes retrieved or in the rates of biochemical pregnancy, clinical pregnancy, spontaneous miscarriage, and live birth between the groups. CONCLUSION: Our findings suggest that compared to OCPs, pretreatment with transdermal E₂ is associated with a shorter duration of ovarian stimulation and lower gonadotropin utilization, without compromising the oocyte yield or pregnancy outcomes in normal-responder patients undergoing fresh IVF.

Effect of Guishen Pill on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Guishen Pill (GSP) on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve (DOR).</p><p><b>METHODS</b>Totally 40 female C57BL/6J mice were randomly divided into 4 groups, the normal control group, the model group, the GSP group, and the dehydroepiandrosterone (DHEA) group, 10 in each group. Pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG), and prostaglandin F2α (PGF2α) were sequentially administrated to produce superovulation. The DOR model was established by exposing to ozone inhalation. Mice in the GSP group were intragastrically administered with GSP at 0.3 mL. Those in the DHEA group were intragastrically administered with DHEA at 0.3 mL. Equal volume of normal saline was intragastrically administered to mice in the normal control group and the model group. All mice wer treated for 21 days. Serum levels of estrogen (E2), progestogen (P), and anti-Müllerian hormone (AMH) were measured by ELISA. Changes of Oct-4, anti-AMH, and early growth response gene-1 (Egr-1) mRNA in ovaries were dtected by Real-time PCR.</p><p><b>RESULTS</b>Compared with the model group, serum levels of E2, P, and AMH, as well as contents of estrogen receptor (ER), progestogen receptor (PR), MVH, and Oct-4 mRNA significantly increased in the GSP group and the DHEA group (P < 0.05).</p><p><b>CONCLUSION</b>GSP could improve expression levels of Oct-4, MVH, and Egr-1 mRNA in DOR mice and their ovarian function.</p>

In vitro fertilization (IVF) in mammals: epigenetic and developmental alterations: scientific and bioethical implications for IVF in humans

The advent of in vitro fertilization (IVF) in animals and humans implies an extraordinary change in the environment where the beginning of a new organism takes place. In mammals fertilization occurs in the maternal oviduct, where there are unique conditions for guaranteeing the encounter of the gametes and the first stages of development of the embryo and thus its future. During this period a major epigenetic reprogramming takes place that is crucial for the normal fate of the embryo. This epigenetic reprogramming is very vulnerable to changes in environmental conditions such as the ones implied in IVF, including in vitro culture, nutrition, light, temperature, oxygen tension, embryo-maternal signaling, and the general absence of protection against foreign elements that could affect the stability of this process. The objective of this review is to update the impact of the various conditions inherent in the use of IVF on the epigenetic profile and outcomes of mammalian embryos, including superovulation, IVF technique, embryo culture and manipulation and absence of embryo-maternal signaling. It also covers the possible transgenerational inheritance of the epigenetic alterations associated with assisted reproductive technologies (ART), including its phenotypic consequences as is in the case of the large offspring syndrome (LOS). Finally, the important scientific and bioethical implications of the results found in animals are discussed in terms of the ART in humans.

The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification / 대한생식의학회지

OBJECTIVE: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. METHODS: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. RESULTS: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). CONCLUSION: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.

Redução da resposta superovulatória de fêmeas bovinas superestimuladas com FSH em doses split / Reduced superovulatory response of bovine females overstimulated with FSH in split doses

Objetivou-se avaliar a superovulação (SOV) de vacas zebuínas, utilizando protocolo convencional ou protocolo com número menor de aplicações e similar dosagem (dose split). Utilizaram-se 16 fêmeas (total 32 SOV), com idade entre 17-42 meses e escore de condição corporal 2,5-4 (escala de 1-5), em delineamento tipo cross-over. No início do tratamento (D0), os animais receberam um dispositivo de progesterona e 2mg de benzoato de estradiol. As fêmeas do grupo convencional receberam 250UI de FSH/LH divididas em oito doses decrescentes administradas em intervalos de 12h (FSH/LH no D4, D5, D6 e D7 no período da manhã e tarde, nas respectivas dosagens: 50,0 UI; 37,5 UI; 25,0 UI; 12,5 UI). No D7 pela manhã, as fêmeas foram tratadas com 150μg de D+cloprostenol, e a remoção da progesterona foi realizada no D7 à tarde. As fêmeas do grupo split também receberam 250 UI de FSH/LH. No D4 de manhã, administraram-se 62,5 UI de FSH/LH via IM e 125 UI por via SC. Quarenta e oito horas após (D6) administraram-se 62,5 UI via SC e na manhã do D7 foi removida a progesterona e aplicaram-se 150μg de D+cloprostenol. As fêmeas de ambos os grupos receberam 50μg de análogo de GnRH no D8 pela manhã e foram inseminadas 12 e 24 horas após. No D15 realizou-se a colheita dos embriões em ambos os tratamentos. Avaliou-se a resposta superovulatória pela contagem do número de folículos e CLs de cada ovário, com auxílio de ultrassom. Todas as variáveis foram submetidas ao teste T de Student para amostras pareadas. Houve diferença (P<0,05) na quantidade de folículos acima de 8mm no D8 (9,06±4,54 e 5,50±4,59); número de CLs no dia da colheita (8,12± 3,26 e 4,69±3,46), número de embriões totais (6,69±3,05 e 3,37±2,50) e de embriões viáveis (5,25±2,29 e 2,37±1,78) nas vacas do grupo convencional em relação às do split, respectivamente. Conclui-se que o protocolo split tem pior resposta superovulatória e de produção in vivo de embriões, em vacas zebuínas, quando comparado ao protocolo convencional...

The aim of this study was to evaluate the superovulation (SOV) response of zebu cows, using conventional protocol or other protocol with similar dose but smaller number of applications (split dose). 16 females (32 SOV), aged 17-42 months, and body condition score of 2.5-4 (1-5 scale) were used in randomized cross-over. At the start of treatment (D0), the animals received progesterone device and 2mg of estradiol benzoate. The females from the conventional group received 250 IU of FSH/LH divided into eight decreasing doses administered at intervals of 12 hours (FSH/LH in D4, D5, D6 and D7, with their respective strengths: 50.0 IU, 37.5 IU, 25.0 IU, 12.5 IU). In D7, the females were treated with 150μg of D+cloprostenol, and the removal of progesterone device was held in the afternoon. The females from the split group also received 250 IU of FSH/LH. In the morning D4 was administered in 62.5 IU FSH/LH IM and 125 IU subcutaneously. Forty eight hours later (D6) 62.5 IU was administered subcutaneously in the morning and on D7, the progesterone device was removed and 150 μg of D+cloprostenol was applied. The females in both groups were given 50 μg of GnRH in the morning and on D8 were inseminated after 12 and 24 hours. On D15 the embryo collection was performed in both treatments. The evaluation of superovulatory response was done by counting the number of follicles and corpus luteum (CL) in each ovary, with the aid of ultrasound. The variables were assessed by Student's t test for paired samples...

Younger women with ovulation disorders and unexplained infertility predict a higher success rate in superovulation (SO) intrauterine insemination (IUI)

<p><b>INTRODUCTION</b>Superovulation-intrauterine insemination (SO-IUI) is the most common assisted reproductive technique (ART) in the world, with good evidence of efficacy and cost-effectiveness. However, parameters affecting its success have not been consistently reported. So in this study, we aim at determining the parameters influencing the success rate of SO-IUI.</p><p><b>MATERIALS AND METHODS</b>We conducted a retrospective cohort study of 797 SO-IUI cycles from 606 patients, performed between 2007 and 2009 in a single centre. These women received clomiphene citrate (CC), recombinant FSH (rFSH) or both.</p><p><b>RESULTS</b>There were 127 clinical pregnancies with a pregnancy rate (PR) of 15.9% (127/797) per treatment cycle. Factors associated with higher PR included maternal age <38 (P = 0.02), subfertility diagnoses of ovulatory disorders, unexplained infertility, sexual dysfunction and unilateral tubal obstruction (P = 0.02), an endometrial thickness ≥8 mm (P = 0.03), total number motile spermatozoa (TNMS) of ≥1 million (P = 0.03), and spermatozoa normal forms (NF) ≥4% (P <0.01) on bivariate analysis. When CC is used, the endometrial thickness is more likely to be suboptimal (<8 mm). All the above parameters remained significant except the subfertility diagnoses on multivariate analysis.</p><p><b>CONCLUSION</b>Patients' selection with women <38 years old and preferably with ovulation disorders and unexplained infertility is associated with the highest PR in SO-IUI. Cycle parameters such as the use of rFSH alone, with the avoidance of CC, TNMS ≥1 million and NF ≥4% is likely to result in the best outcomes and reduce the high order multiple pregnancy risk.</p>

The effect of various assisted hatching techniques on the mouse early embryo development / 대한생식의학회지

OBJECTIVE: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers. METHODS: Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed. RESULTS: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05). CONCLUSION: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.

Effect of superovulation on uterine and serum biochemical parameters and its potential association with transferable embryos in Holstein dairy cows

The objective of this study was to determine the effects of superovulation (SOV) on serum and uterine biochemical parameters, uterine bacteriology and cytology and number of transferable embryos (TE). Dairy cows were placed on a Presynch/CIDR Synch protocol. The SOV group was superovulated, induced in estrus, and inseminated, whereas the control group was induced in estrus and inseminated without SOV. Uterine bacteriology and cytology and uterine and serum biochemical parameters were measured at day 7 of the estrous cycle to start the SOV protocol, as well as on the day of embryo recovery (DER). The SOV group produced 7.5 +/- 6.7 oocytes/embryos, of which 3.4 +/- 4.7 were TE. Serum urea and E2 and uterine Glu, CK, LDH, TP, P4 and PGFM in the control group and serum P4 and PGFM and uterine LDH and PGFM in the SOV group were significantly higher (p < 0.01) at DER than day 7. At DER, uterine urea, LDH, PGFM and TP and serum urea, LDH, PGFM, and P4 concentrations were higher (p < 0.01) in the SOV group than the control. There was no significant variation in uterine bacteriology or cytology. Overall, these results infer that SOV affects both serum profile and uterine secretions, and that these changes may influence the number of TE.

Morfometria do aparelho genital e resposta superovulatória de coelhas suplementadas com geleia real / Morphometry of the genital tract and superovulatory response in rabbits supplemented with royal jelly

Avaliou-se o efeito da suplementação com geleia real sobre a morfometria do aparelho genital, resposta superovulatória e qualidade embrionária de coelhas. Trinta e seis fêmeas foram distribuídas em quatro grupos (G), sendo: G1 (n=9) formado por animais não suplementados com geleia real, e G2, G3 e G4 (n=9 em cada grupo) por animais suplementados com 10, 20 e 40mg/dia de geleia real. A superovulação consistiu na aplicação de 40UI de gonadotrofina coriônica equina, seguida por 40UI de gonadotrofina coriônica humana, via intramuscular, 48 horas após, e submetidas à cobrição natural. Os animais foram sacrificados, e os embriões coletados 72 horas após a cópula. Não houve diferença estatística entre tratamentos para as variáveis analisadas. O peso médio do aparelho genital foi de 10,88±0,38g; dos ovários - direito e esquerdo -, 0,28±0,02g; e o índice gonadossomático, 0,02±0,0g. O número médio de estruturas totais recuperadas foi de 9,2±1,4; de embriões viáveis, 8,7±1,4; e de degenerados, 0,5±0,2. Dos embriões viáveis, 5,6±0,8 foram classificados como grau I; 2,3±0,5, como grau II; e 0,8±0,2, como grau III. A suplementação com geleia real na dose de até 40mg/dia não apresentou efeito estimulador sobre o aparelho genital e a qualidade embrionária de coelhas.

The purpose of this paper was to assess the effect of supplementation of royal jelly on the morphometry of the genital tract, superovulatory response and embryonic quality in adult rabbits. 36 rabbits were distributed in four groups (G), as follows: G1 (n=9): no supplementation with royal jelly, G2, 3 and 4 (n=9): supplementation with 10, 20 and 40mg/day of royal jelly. The superovulatory protocol consisted in the application of a dose of 40UI of Equine Chorionic Gonadotrophin, followed by a dose of UI of Human Chorionic Gonadotrophin, intramuscularly, 48 hours later, and submitted to natural cover. The animals were euthanasiated and the embryos collected 72 hours after copulation. There was no statistical difference for the analyzed variables (P>0.05). The average weight for the female genital tract was 10.89±0.38g; the average weight of right and left ovaries was 0.28±0.02g and the average gonadosomatic index rate was 0.02±0.0g. The number of total recovered structures was 9.2±1.4. The average number of viable embryos was 8.7±1.4 and of degenerated embryos was 0.5±0.2. Viable embryos were morphologically classified as degree I: 5.6±0.8; degree II: 2.3±0.5; degree III: 0.8±0.2. Royal jelly did not present stimulatory effect on organs related to the reproduction of rabbits, nor was it effective to improve their embryonic quality at dosages of up to 40mg of in natura royal jelly.

Intervention of controlled ovarian hyperstimulation cycle by Chinese medical herbs for Shen tonifying, blood nourishing and activating: a randomized clinical trial / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Chinese medical herbs for Shen tonifying, blood nourishing and activating (CMHSTBNA) on the cycle of controlled ovarian hyperstimulation (COH) of assisted reproductive technique (ART).</p><p><b>METHODS</b>A large sample randomized control trial was performed. Infertility women patients, younger than 42 years (infertility due to tubal factor and/or male factor) were randomly assigned to the CMHSTBNA intervention group (abbreviated as the treated group) and the control group, 184 cases in each group. All underwent COH. Those in the treated group received assist therapy of CMHSTBNA from the menstrual period day 2 -3 of COH to the day of oocytes retrieved. The serum hormone level [including estrogen (E2), progesterone(P), luteal hormone (LH) on the day of human chorionic gonadotrophin (hCG) administration], the medication days and dosage of gonadotropin (Gn), the number of oocytes retrieved, the fertilization rate, and the good-quality embryo rate were observed and compared with the control group.</p><p><b>RESULTS</b>The endometrial thickness on the day of oocytes retrieved was 10.85 +/- 1.63 mm in the treated group, larger than that in the control group (10.50 +/- 1.49 mm) (P <0.05). The good-quality embryo rate and the frozen rate were 48. 9% and 39. 7% respectively in the treated group, superior to those of the control group (45. 4% and 35. 8% respectively), showing statistical difference (P < 0.05). On the day of hCG administration, favorable tendency was shown in the serum levels of estradial (E2), progesterone (P), luteinizing hormone (LH), the medication days and dosage of Gn, the number of oocytes retrieved, the fertilization rate, and the cleavage rate, showing no statistical difference when compared with the control group (P >0.05).</p><p><b>CONCLUSION</b>The combined application of CMHSTBNA and gonado-trophic hormones in COH cycle could elevate the embryo quality, improve the endometrial state, thus laying foundation for successful in vitro fertilization/intracytoplasmic sperm embryo transfer.</p>

Ultrastructural and morphometrical study of preimplantation endometrium in superovulated mice treated with progesterone or sildenafil

Endometrial development has an important role in blastocyst adhesion and implantation. During IVF cycles, endometrial development is enhanced by progesterone. The aim of this study was to compare ultrastructural and morphometrical characteristics of mice uterine endometrium in natural cycle with those in superovulated cycles received progesterone or Sildenafil. In This study, 60 female bulb/c mice were divided into 4 groups: a control and 3 experimental; gonadotropin, gonadotropin+ Sildenafil and gonadotropin+ progesterone. In experimental groups the mice superovulated mated. In the gonadotropin+ progesterone and gonadotropin+ Viagra groups, the mice respectively received 1mg progesterone and 3 mg Sildenafil citrate. Their uterine specimens were prepared for morphometrical and ultrastructural study. Height of the epithelial cells was measured, using motic software. Statistical analysis was performed using ANOVA. Microscopy revealed that in control group the cells had numerous apical microvilli and the height of the cells was 20.52 +/- 2.43 microm. In gonadotropin+ progesterone group, the granules were found in basal and apical portions and cellular height were 17.91 +/- 2.78 microm which were significantly shorter than in the control and gonadotropin groups [p<0.001]. In this group, the apical membrane also contained pinopodes. In gonadotropin +Sildenafil group, the granules were found in both apical and basal portions and the height of the cells were 17.60 +/- 2.49 microm which were significantly shorter than in the control and gonadotropin groups [p<0.001]. In this group, pinopodes appeared slightly extensive than the other groups. It is concluded that superovulatory drugs in mice stimulate endometrial maturation but injection of Sildenafil is nearly more positive