Optimization of superovulation in the reproductively mature mouse

Optimum gonadotropin doses and chronology were established for the induction of superovulation in sexually mature hybrid mice (BALB/cBy×C57BL/6By). A regime of 12 IU pregnant mares' serum gonadotropin (PMSG), followed 48 hr later by 20 IU human chorionic gonadotropin (hCG) administered 1 hr before the midpoint of the light cycle (1200), gave the maximum ovulatory response. There was no evidence that endogenous luteinizing hormone influenced the superovulation response to exogenous gonadotropins. Fewer than 50% of zygotes reached the blastocyst stage (90–93 hr post hCG), with the greatest rate of loss at the two-to four-cell stage. Litter size following superovulation was 19.6±0.9. There was no significant difference between the number of blastocysts observed and litter size. Similarly, counts of mature follicles in ovaries prior to hCG stimulation were not significantly greater than the number of secondary oocytes that subsequently ovulated. These data indicate that standard superovulation protocols may require finetuning to maximize productivity and confirm that embryo loss is greatest between the first cleavage division and blastocyst formation.     

In vitro preimplantation mouse embryo development with incubation temperatures of 37 and 39°C

Embryos from two strains of mice were used to assess the effect of incubation temperature on pronuclear and twocell development to the morula/blastocyst (M/B) stage. Embryos from B6D2F2 and B6SJLF1 strains were cultured in medium M16 at either 37 or 39°C until 120 hr post human chorionic gonadotropin (hCG) or 0, 24, or 48 hr at 37°C and the remaining time at 39°C. Overall M/B development for pronuclear embryos was 0.6, 0, 32.3, and 52.4% for 0—96, 24—72, 48—48, and 96—0 hr at 37 and 39°C, respectively. Only 0—96 and 24—72 hr at 37 and 39°C were not different (P >0.10). Overall M/B development for two-cell embryos was 48.1, 78.1, and 98.0% for 0—72, 24—48, and 72—0 hr at 37 and 39°C, respectively. Percentage development at each time was different (P <.01) for each category. Additionally, the number of nuclei for morulae and blastocysts tended to be higher for embryos initiating culture at the two-cell stage compared to pronuclear embryos. The first cell cycle was most dramatically affected by a 2°C increase in incubator temperature. More advanced embryos can tolerate slight increases in incubator temperature more readily than pronuclear embryos.     

Recombinant-human luteinizing hormone (r-hLH) as ovulatory stimulus in superovulated does

Purpose To study the effects of r-hLH as ovulatory stimulus in does.Method New Zealand does, 18 kw old, in estrus, received 25 IU of pregnant mare serum gonadotropin (PMSG) followed at 48 h either by 50 IU of r-hLH (n=20) or hCG (n=20) to induce follicular growth and ovulation. All does were previously artificially inseminated to avoid endogenous LH surge. Half of the animals receiving r-hLH (n=10) or hCG (n=10) were killed at 72 h after the hormone administration, and the remaining half were killed at 14 days. At 72 h the number of corpora lutea and preovulatory folliclés was determined, and fertilization rate, embryo quality, degree of embryonic development, and oviductal transit were all assessed. On Day 14 the number corpora lutea and implanted embryos were counted, and implantation rate was determined. Median and interquartile ranges were calculated for each parameter.Results At 72 h the median for corpora lutea was 8 (7–10) in the r-hLH group vs 13 (10–14) in the hCG group (P=0.009); preopvulatory follicles were 7 (6–10) vs 0 (0-0) (P=0.0007); the percentage of good-quality embryos was 71.4% (54.5–75) vs 33.3% (25–37.5) (P=0.001), for intermediate-quality embryos it was 25% (14.3–36.4) vs 33.3% (25–38.5), and the percentage of degenerated embryos was 0% (0–12.5) vs 33.3% (25–37.5) (P=0.015), respectively. Fertilization rates were similar in both groups. Embryonic development was more homogeneous in the animals receiving r-hLH (8 to 16 cells) compared to those receiving hCG (2 to 16 cells). The median of embryos still in oviducts at 72 h was significantly higher in the hCG group [6 (4–13)] than in the r-hLH group [0 (0–4)] (P=0.41). At 14 days the median of corpora lutea was higher in the hCG [12 (11–16)] than in the r-hLH group [10(7–13)] (P=0.008), but no differences were noted in the number of implanted embryos. Implantation rate was higher in the r-hLH group [100 (92.3–100)] than in the hCG group [87.5 (83.3–94.1)] (P=0.056).Conclusions At the studied dose an ovulatory stimulus with r-hLH induced fewer follicles to ovulate than hCG. Recombinant-hLH produced superior embryo morphological quality, a more homogeneous degree of embryo development, and more synchronous embryo transit than hCG. In spite of the larger number of ovulations following hCG, subsequent events essential for pregnancy were higher with r-hLH, offsetting differences in terms of implanted embryos at 14 days of pregnancy.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Impact of ketorolac administration around ovarian stimulation on in vivo and in vitro fertilization and subsequent embryo development

Abstract

We performed this study to investigate the effect of ketorolac (a non-steroidal anti-inflammatory drug) administration around ovarian stimulation on in vivo and in vitro fertilization process. Sixty-four female mice (ICR) were injected with ketorolac (0, 7.5, 15 and 30?µg/d) for 3?d starting from the day of eCG treatment. In experiment 1, 41 mice were triggered by hCG and then mated; two-cell embryos were obtained and in vitro development up to blastocyst was observed. In experiment 2, 23 mice were triggered by hCG and mature oocytes were collected; in vitro fertilization rate and subsequent embryo development up to blastocyst was recorded. In experiment 1, the blastocyst-forming rates per in vivo fertilized two-cell embryo showed an inverse relationship with a dosage of ketorolac (97.6%, 64.2%, 35.4% and 25.9%). In experiment 2, degenerated oocytes were frequently observed in a dose-dependent manner (4.3%, 22.9%, 22.4% and 75.0%). Lower fertilization rates were noted in all the three ketorolac-treating groups; blastocyst-forming rate was significantly lower in 30-µg-treating group when compared with the control group. Administration of ketorolac around ovarian stimulation significantly affects the development of in vivo fertilized embryo in a dose-dependent manner. High-dose ketorolac could result in a poor oocyte quality and decreased embryo developmental competence.     

Sequential observation of mitochondrial distribution in mouse oocytes and embryos

Objective The purpose of this study was to elucidate changes in the distribution of mitochondria through the cell cycle.Materials and Methods Mouse oocytes and embryos were recovered sequentially from mice and stained with the vital fluorescent mitochondrial stain rhodamine 123. Mitochondrial staining pattern were classified into three types: aggregation (Ag), homogeneous (H), and perinuclear accumulation (PA).Results Sequential observations revealed that mitochondria of oocytes and embryos grown in vivo translocated in the cytoplasm during the cell cycle, showing the H pattern be fore human chorionic gonadotropin (hCG) administration, the PA pattern 8–9 hr post-hCG, the H pattern again 10–14 hr post-hCG, and the PA pattern again 24 and 31–32 hr post-hCG following fertilization. In the twocell stage, the Ag pattern was shown 35 hr post-hCG, the H pattern was observed 40 hr post-hCG, and the PA pattern was found 48 hr post-hCG. In the embryos cultured in vitro and showing developmental block, mitochondrial translocation was shown to be inhibited after they aggregated in the early two-cell stage (35 hr post-hCG). Moreover, the translocation of mitochondria was restored by the addition of superoxide dismutase or thioredoxin to the culture medium. Both of these enzymes have already been shown to have the ability to overcome developmental block.Conclusion The present study revealed that mitochondria translocated in the cell cycle and suggested that there is a close relationship between mitochondrial translocation and developmental arrest.     

Requirements for Human Chorionic Gonadotropin and Recombinant Human Luteinizing Hormone for Follicular Development and Maturation

Purpose: Our purpose was to evaluate the requirements for human chorionic gonadotropin (hCG) and recombinant luteinizing hormone (rec-LH) for follicular development and maturation in mice. Methods: We carried out ovarian stimulation of immature mice. Output parameters were the preembryos created in vivo and frequency of blastocyst formation in vitro. Results: hCG at 0 to 1 IU resulted in a dose-dependent recovery of preembryos (0 to 39.7 ± 4.3; mean ± SE) per mouse. hCG at 1 and 10 hCG gave similar results, whereas higher doses significantly reduced the number of preembryos. Potential for blastocyst formation was independent of hCG dose. hCG and rec-LH together exerted a synergistic effect on the recovery of preembryos. Conclusions: Optimal follicular development required a combination of 20 IU follicle stimulating hormone and 1–10 IU hCG. The potency of hCG was higher than that of rec-LH, but a synergistic effect of rec-LH and hCG was observed. The results may be pertinent for the development of strategies for ovarian stimulation of women with low levels of endogenous LH.     

The role of a human chorionic gonadotropin burst in in vitro fertilization

Findings: No oocytes were found during four ovum pickups (OPU), despite a satisfactory ovarian response to controlled ovarian hyperstimulation. After the first attempt failed in the fourth case, five eggs were retrieved, fertilized, and cleaved after cycle rescue with hCG. Conclusions: Whenever oocytes are not aspirated during OPU due to a lack of hCG administration, the cycle may be rescued if 10,000 IU of hCG is injected immediately and OPU planned for 33–36 hr later.     

Effects of high-dose and multiple-dose gonadotropin stimulation on mouse oocyte quality as assessed by preimplantation development following in vitro fertilization

The effects of increasing the level of ovarian stimulation on preimplantation embryonic development were assessed using a mouse in vitro fertilization system. When F₁ hybrid (C57BL/6 × CBA/Ca) mice received a single injection of 5 IU pregnant mare's serum gonadotropin (PMSG) followed 60 hr later by 5 IU human chorionic gonadotropin (hCG) approximately 50% of the resultant postovulatory oocytes developed to the blastocyst stage following in vitro fertilization. Increasing the single dose of PMSG to 10 or 15 IU resulted in significant reductions in the frequency of development to the blastocyst stage. When one or two additional doses of 5 IU PMSG were administered 24 and 48 hr after an initial injection of 5 IU, lower frequencies of oocytes with the potential for full preimplantation development were again observed. This reduction in gamete quality was significantly greater when the final dose of PMSG was administered only 12 hr prior to hCG. The results suggest that excessive gonadotropin stimulation may compromise the quality of the preimplantation embryos obtained following in vitro fertilization and that the timing of gonadotropin administration may also be critical.     

Twin pregnancy obtained with frozen-thawed embryos after in vitro maturation in a patient with polycystic ovarian syndrome

Purpose : A twin pregnancy was obtained in a patient with polycystic ovary syndrome after the transfer of three in vitro maturation-derived day 3 embryos that has been frozen and thawed. Methods : The patient had received mild hMG stimulation followed by hCG injection. After culture for 24–48 h, mature oocytes were fertilized by ICSI. Embryos were cultured until day 3; supernumerary embryos were cryopreserved using a slow protocol. Results : Among 15 nonatretic oocytes, 9 matured, 8 were fertilized. Four embryos were transferred but they did not implant. The subsequent transfer of three frozen–thawed embryos resulted in the delivery of two healthy girls. Conclusions : These results indicate that a pregnancy could be obtained with in vitro maturation-derived day-3 frozen–thawed embryos.     

Effect of anordrin on the development of mouse preimplantation embryos in vitro

Purpose: The in vitro effect of anordrin and anordiol on the development of mouse two-cell embryos was studied. Method: Female mice were primed with gonadotropins for superovulation and caged with male mice. Preimplantation embryos, at the two-cell stage, were recovered from the oviducts at 40 hr post-hCG. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of anordrin for 3, 12, 24, and 80 hr and then grown in the anordrin-free culture medium and assessed for the formation of total and hatching blastocysts at 80 hr. In the second experiment, two-cell embryos were grown in culture medium containing different concentrations of anordiol and assessed for the formation of total and hatching blastocysts at 80 hr in vitro. Results: Exposure of two-cell embryos to anordrin concentrations of 2.5–7.5µg/ml for 12 hr, 2.5–5.0µg/ml for 24 hr, and 2.5µg/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 2.5–7.5µg/ml for 12 hr, 1.0–2.5µg/ml for 24 hr, and 1.0µg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts, in a exposure time-dependent and dose-dependent manner. Exposure of two-cell embryos to anordiol concentrations of 15–25µg/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 15–20µg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner. Conclusion: Anordrin and its metabolite anordiol inhibit the development of two-cell embryos in vitro.Presented orally at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994, Abstract No. O-176.     

Superovulation alters DNA methyltransferase protein expression in mouse oocytes and early embryos

Purpose

DNA methylation is an epigenetic mechanism that plays critical roles during mammalian oocyte and preimplantation embryo development. It is achieved by adding a methyl group to the fifth carbon atom of cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinucleotide sites using DNA methyltransferase (DNMT) enzymes for de novo and maintenance methylation processes. DNMT1, DNMT3A, and DNMT3B play important roles in establishing methylation of developmentally related genes in oocytes and early embryos. The purpose of this study is to identify the effect of superovulation on the expression and subcellular localizations of these three DNMT enzymes in the mouse oocytes and early embryos.

Methods

Three groups composed of control, normal dose [5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG)], and high dose [7.5 IU PMSG and 7.5 IU hCG] were created from 4–5-week-old female BALB/c mice. The relative expression and subcellular localizations of the DNMT proteins in the control and experiment groups have been characterized by using immunofluorescence staining subsequently analyzed in detailed.

Results

DNMT1, DNMT3A, and DNMT3B protein expression in the germinal vesicle and metaphase II oocytes and in one-cell and two-cell embryos differed significantly when some of the normal- and high-dose groups were compared with the control counterparts.

Conclusion

This study has demonstrated for the first time that superovulation alters expression levels of the DNMT proteins, a finding that indicates that certain developmental defects in superovulated oocytes and early embryos may result from impaired DNA methylation processes.

     

体外加入hCG不能改善人卵子体外成熟及发育潜能

目的:研究体外培养过程中hCG对人未成熟卵子体外成熟和发育潜能的影响。方法:62例PCOS不孕患者进行了89个未成熟卵子体外成熟培养(IVM)周期,根据体外成熟培养液中有无hCG,将其分为A组(29个周期)采用常规IVM培养液培养;B组(30个周期)先在去除hCG的常规IVM培养液中培养10h,然后改在常规IVM培养液中培养;C组(30个周期)在去除hCG的常规IVM培养液中培养。所有卵子体外培养24-48h,成熟后卵子分批行单精子注射授精(ICSI),培养2-3d后进行胚胎移植。结果:A、B、C组32h、48h卵子体外成熟率分别为46.02%,69.25%;43.72%,64.51%;51.87%,67.51%;组间无显著差异。受精率、卵裂率、临床妊娠率及种植率各组间也均无显著差异(P>0.05)。结论:对于PCOS患者,有无hCG对卵子体外成熟、胚胎发育及临床结局均没有显著影响。     

Effects of passive immunization against estradiol on rabbit ovum transport

The role played by estradiol in control of ovum transport was studied in rabbits that were induced to ovulate by hCG stimulation. Withholding estrogen from target tissues during ovum transport by passive immunization with sheep anti-estradiol immunoglobulin (AE) resulted in accelerated oviductal transport and expulsion of ova from the uterus. The degree of accelerated transport was dependent on the duration of AE treatment. When AE treatment was started 24 h before hCG, fewer ova were recovered from the reproductive tracts at 72 h after hCG than were recovered from tracts of animals treated with a nonspecific immunoglobulin; the location of ova at 48 h after hCG was unaltered by this AE regimen. When AE treatment was started 72 h before hCG, fewer ova remained in the oviducts than were found in the tubes of controls at 48 h after hCG; and at 48 h after hCG, ova were found in the uteri of animals that had been treated with AE starting 72 h before hCG. When AE treatment was started 48 h before hCG, the position of eggs within the reproductive tract was not different from controls at 48 h after hCG. These observations support the concept that estrogen withdrawal is involved in the transport of ova through the oviduct of the rabbit, and suggest that the lack of estrogen secretion from the time of ovulation through the transport period is important in the control of normal ovum transport.     

Neem oil inhibits two-cell embryo development and trophectoderm attachment and proliferation in vitro

Purpose The in vitro effect of neem oil was studied on the development of mouse two-cell embryos and trophectodermal cell attachment and proliferation.Method Female mice were primed with gonadotropins for superovulation and caged with male mice. Early embryos, at the two-cell and the blastocyst stages, were recovered at 40 and 88 hr post-hCG from the oviducts and the uteri, respectively. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of neem oil for 1, 12, and 24 hr and then grown in neem oil-free culture medium and assessed for the formation of total and hatching blastocysts at 96 hr. In the second experiment, partially hatching blastocysts were cocultured with human endometrial stromal cell monolayers in culture medium containing different concentrations of neem oil and assessed for the attachment and proliferation of trophectodermal cells at 96 hr.Results Exposure of two-cell embryos to neem oil concentrations of 0.050–0.500% for 1 hr, 0.010–0.250% for 12 hr, and 0.005–0.100% for 24 hr caused significant inhibition of the formation of total and hatching blastocysts, in a dose-dependent manner. Neem oil at 0.050–0.100% concentrations inhibited, in a dose-dependent manner, the in vitro attachment and proliferation of trophectodermal cells of partially hatching blastocysts cocultured with human endometrial stromal cells monolayers.Conclusion Neem oil inhibits the development of two-cell embryos and attachment and proliferation of the trophectodermal cells of partially hatching blastocysts in vitro. The study encourages the use of this herbal product as a postcoital contraceptive that warrants further research.Presented at the 41st Annual Meeting of the Society for Gynecologic Investigation, Chicago, Illinois, March 22–26, 1994, Abstract No. P243.     

Preimplantation development following in vitro fertilization of mouse oocytes: Effects of timing of superovulation and preincubation in vitro

The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F₁ hybrid C57BL/6×CBA/Ca mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.     

Successful delivery following cryopreservation of zygotes produced by in vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome-like disease: a case report

Purpose : To estimate frozen zygotes, which developed from in vitro matured oocytes retrieved from polycystic ovarian syndrome-like disease. Methods : Oocyte retrieval was performed on Day 15 following withdrawal bleeding. The oocytes were incubated for 24 h in TCM-199 maturation medium supplemented with follicle fluid, E2, FSH, and hCG. Results : A total of 12 immature oocytes were collected. Seven of the 12 oocytes (58.3%) developed to the metaphase-II stage, and subsequently, all seven fertilized oocytes were frozen at the pronuclear stage. The remaining five oocytes failed to develop to the metaphase-II stage after an additional 24 h of incubation. Three of seven cryopreserved oocytes were thawed and developed to 2–8-cell cleaved stage embryos. The first pregnancy failed. However, the second frozen–thawed embryo transfer resulted in the delivery of healthy twins. Conclusions : Successful delivery using frozen zygotes from an anovulatory woman with polycystic ovarian syndrome-like disease.     

A prospective,randomized and blinded comparison between 10,000 IU urinary and 250 μg recombinant human chorionic gonadotropin for oocyte maturation in in vitro fertilization cycles

Purpose: To compare the efficacy and safety of u-hCG with r-hCG in IVF cycles.Methods: A prospective, investigator-blind, randomized, comparative study. Patients (n = 100) ≤ 35 years with IVF indication were randomly assigned on the day of hCG administration for oocyte maturation to receive either u-hCG (10,000 IU) or r-hCG (250 μ g).Results: No statistical differences were found between groups in relation to total number of oocytes retrieved, percentage of mature oocytes, number of injected oocytes, fertilization rates and number of embryos transferred. The data indicate a possible trend toward a higher incidence of pregnancy in the r-hCG group. Adverse events, predominantly injection-site reactions, were significantly more common in the u-hCG group.Conclusions: r-hCG is at least as effective for inducing final stages of oocyte maturation as 10,000 IU u-hCG and is also associated with significantly better patient tolerance and thus higher patient acceptability.     

Maternal peripheral T helper 1-type and T helper 2-type immunity in women during the first trimester of twin pregnancy

Introduction This study investigated the T helper Th1:Th2 balance in twin pregnancies compared with singleton pregnancies during the first trimester.Methods Blood samples were taken from 24 women with a singleton pregnancy and 14 women with twin pregnancy at 8–9 weeks gestation to examine the ratios of Th1:Th2 and serum human chorionic gonadotropin (hCG) and progesterone levels.Results The average ratio of Th1:Th2 in the twin pregnancies was significantly lower than that in singleton pregnancies (7.3±2.3 vs. 10.5±2.2, p<0.05). There were negative correlations between the Th1:Th2 ratio and serum hCG levels (mIU/ml) (Th1:Th2 ratio = 14.5–4.52×10^–5×hCG, r²=0.41, p<0.05) and between the Th1:Th2 ratio and serum progesterone levels (ng/dl; Th1:Th2 ratio = 23.0–0.63 × progesterone, r²=0.36, p<0.05).Conclusion Our findings show marked predominance of Th2 type cytokines occurring in twin pregnancies is related to the increase in trophoblasts during the first trimester.     

Influence of human chorionic gonadotropin (hCG) and hCG internalization by granulosa cells on the rate of in vitro fertilization and embryonic development of human oocytes

Follicular fluids and granulosa cells were obtained from 28 aspirated follicles of nine women undergoing laparoscopy in an in vitro fertilization program. Follicular growth was stimulated by a human menopausal gonadotropin regimen and laparoscopy was performed 32 hours after human chorionic gonadotropin (hCG) administration. Follicular fluid 17 beta-estradiol (E2) levels were higher and hCG levels were lower in follicles with oocytes that fertilized and cleaved beyond two blastomeres (greater than two-cell group) than in those with nonfertilizable oocytes (NF group) (P less than 0.05). Compared to those from the NF group, granulosa cells from the greater than two-cell group secreted less progesterone (P) in vitro and had a fourfold increase in percentage of cells with internalized hCG. These results demonstrate that the steroidogenic capacity of granulosa cells from follicles whose oocytes fertilize and undergo accelerated embryonic development in vitro differs from the capacity of granulosa cells from NF follicles. This difference may be due to their enhanced ability to bind and subsequently internalize hCG.     

In vitro maturation and fertilization of oocytes isolated from aged mice: A strategy to rescue valuable genetic resources

Purpose This project was to determine whether oocytes isolated from virgin aged mice, up to 18 months old, are competent to undergo cytoplasmic maturation in vitro and undergo fertilization and embryonic development. If so, oocyte maturation in vitro could be used as a strategy to rescue valuable genetic resources.Results Although the number of oocytes recovered from mice was greatly reduced with increasing age, the percentage of oocytes that underwent fertilization, cleavage, and development to the blastocyst stage was essentially unchanged up to 18 months of age. The success of cleavage to the two-cell stage was greater after maturation in vitro (81%) than gonadotropin-induced maturation in vivo (55%). About 20% (20/106) of the embryos derived from oocytes isolated from 18-month-old mice developed to term after embryo transfer.Conclusion Oocytes from virgin aged mice undergo normal cytoplasmic maturation in vitro. Higher percentages of oocytes from aged mice cleave to the two-cell stage after spontaneous maturation in vitro than after gonadotropin-induced maturation in vivo. Therefore, in vitro maturation and fertilization of oocytes could be used to rescue valuable genetic resources that might otherwise be lost because of age-related infertility.