Genotoxic and embryotoxic effects of gonadotropin-hyperstimulated ovulation of murine oocytes, preimplantation embryos, and term fetuses.

Compared to spontaneous ovulation, gonadotropin-hyperstimulated ovulation (superovulation) in mice resulted in a fourfold increase in the number of preimplantation embryos 3 days post coitum, 50% of which died before term. Both in vitro development of embryos during the preimplantation period and transfer of morulae from superovulated females to pseudopregnant untreated foster mothers indicate that the prenatal loss occurring shortly before implantation up to term is due to maternal factors rather than to direct hormonal effects on oocytes or early embryos. Indeed, no genotoxic events could be observed in 4-cell to blastocyst stage embryos from superovulated female mice as revealed by the chromosomal aberration test and the sister chromatid exchange assay. Chromosome analysis of the pronuclei from mouse zygotes showed an increased rate of aberrations in oocyte-derived nuclei after superovulation in comparison to spontaneous ovulation. The present data suggest that aberrant murine oocytes may be fertilized, but they do not survive the first cleavage stages. The result is discussed with respect to the high incidence of chromosomal abnormalities found in human oocytes after gonadotropin-hyperstimulated ovulation.     

Cytogenetic studies on preimplantation mouse embryos exposed to methylnitrosourea in vivo

Since exposure of mice to methylnitrosourea (MNU) during the preimplantation period can induce malformations and an increased postnatal death rate, direct embryotoxic effects were studied in preimplantation embryos shortly after treatment of pregnant mice on days 2 and 3 of gestation with single i.p. injections of 2.5, 5.0, and 10.0 mg/kg MNU. Embryos exposed to MNU for 24 h after treatment on day 2 showed a significant reduction of cell number and induction of sister chromatid exchange (SCE) frequency, but no structural chromosomal aberrations or inhibition of development during culture. Embryos exposed to MNU in vivo for 3 h on day 3 showed significantly reduced cell numbers, a significant inhibition of development in culture, and an increase in structural chromosome aberrations. Due to the high cytotoxicity of MNU, determination of SCE was not possible. The results indicate that MNU reaches preimplantation mouse embryos shortly after maternal treatment and that malformations seen at term and postnatal effects are probably induced by the direct action of MNU on early embryos. Furthermore, the importance of the time interval chosen for evaluation of toxicologic endpoints in preimplantation embryos is demonstrated.     

Effects of paraquat on development of preimplantation embryos in vivo and in vitro

Paraquat can cause oxidative stress through redox cycling, and preimplantation embryos are sensitive to oxidative stress in vitro. In this study, the effects of paraquat on preimplantation embryo development were examined. Exposure of preimplantation embryos (collected on the day after ovulation) to paraquat in vitro for 24 h at concentrations as low as 8 microM caused a significant decrease in the percentage of 8-cell embryos and an increase in the percentage of compacted morulae, but the content of reduced glutathione (GSH) in embryos was not changed. Altered embryo development was most likely due to premature compaction because a 42% decrease in cell number per compacted morulae was observed in embryos exposed to paraquat at 1 mM. Exposure of preimplantation embryos to paraquat in vitro for 4 days at 200 microM or higher eliminated development beyond the blastocyst stage. Exposure of bred female mice to paraquat at 30 mg/kg on day 2 after ovulation led to a small but significant decrease in the percentage of 8-cell embryos on day 3 without a detectable increase in the percentage of compacted morulae. No detectable change in preimplantation embryo development was found following paraquat exposure on the day of ovulation (day 0), although a significant decrease in embryo GSH was found on day 1. These data indicate that paraquat can adversely impact the development of preimplantation embryos in vitro and in vivo without consistent modulation of GSH level.     

超长降调节超促排卵日促黄体素水平对子宫内膜异位症体外受精结局的影响

目的探讨改良超长降调节方案超促排卵启动日促黄体素(LH)与子宫内膜异位症患者体外受精(IVF)结局的关系。方法 50例子宫内膜异位症患者接受改良超长方案治疗,患者接受性腺素释放激素激动剂(GnRH-a)治疗3～6 mo,每次曲普瑞林1.88 mg肌注,间隔28 d,第2次注射后2 wk开始检测血清CA125,CA125降至18 U.L-1以下后注射曲普瑞林1.88 mg,于末次注射后2～6 wk开始促性腺素超促排卵行体外受精-胚胎移植(IVF-ET)治疗。按超促排卵启动日血清LH水平分为2组:A组25例,LH<0.5 U.L-1;B组25例,LH≥0.5 U.L-1,比较2组IVF-ET结局。结果 A组促性腺素平均用药天数多于B组[(14.71±24.96)d vs.(8.86±1.92)d,P<0.01],A组种植率低于B组(24.56%vs.33.33%,P<0.05)。结论改良超长降调节方案治疗子宫内膜异位症患者启动日LH过低将增加促性腺素用药天数,降低种植率。建议将LH作为超排启动的主要指标。     

基因重组卵泡刺激素促排卵效果的观察

目的比较在体外受精-胚胎移植(IVF-ET)及卵母细胞单精子显微注射(ICSI)中尿促性素(menotropins,HMG)和重组卵泡刺激素的作用。方法采用促性腺激素释放激素激动剂(GnRH-a)方案降调节,月经第3天随机分成两组,给予两种不同的促性腺激素(Gn)促排卵。A组51例,用国产HMG促排卵;B组58例,采用基因重组卵泡刺激素(rFSH)促排卵,至卵泡成熟后,经阴道B超引导下取卵。按本中心常规方法行IVF/ICSI。结果两组平均获卵率、空卵率、优质胚胎数、受精率、卵裂率、临床妊娠率、ICSI后卵子的死亡率差异有统计学意义。结论应用rFSH促超排卵可获得足量的高质量卵子。     

三种子宫内膜准备方案胚胎移植日血清雌孕激素水平、胚胎种植率、临床妊娠率的比较研究

目的探讨超排卵(Super-ovulation)控制性超排卵(controlled ovarian hyperstimulation,COH)和雌孕激素替代治疗(hormone replacement treatment,HRT)三种子宫内膜准备方案对胚胎移植日血清雌孕激素水平、人胚胎种植率及临床妊娠率的影响。方法2004年1月至2005年10月在我中心采用不同治疗方案行IVF-ET的不孕妇女分为3组:超排卵组、控制性超排卵组和雌孕激素替代治疗组。体外受精-胚胎移植按我中心常规方法进行。酶联免疫吸附双抗夹心法(ELASA)检测ET日血清雌孕激素水平。各组IVF ET的实验室和临床资料进行分析比较。结果同期COH组超排卵药物总剂量和获卵数分别为(30.12±10.18)支和(10.35±2.01)个,明显高于超排卵组(8.21±4.19)支和(5.05±3.11)个(P<0.05);COH组胚胎种植率和临床妊娠率分别为21.19%和34.55%,明显低于HRT组(34.9,52.38%)和超排卵组(32.43%,50%)(P<0.05)。COH组ET日E_2水平(825.24±558.95 pg/mL),明显高于超排卵组(395.8±287.27pg/mL)及HRT组(407.72±123.76pg/mL)(P<0.05)。COH组P水平(109.31±82.61 ng/mL)明显高于HRT组(62.63±31.54 ng/mL)(P<0.05),而与超排卯组比较无明显差异(P>0.05)。COH组E_2/P比值(8.34±1.76)明显高于超排卵组(4.51±2.32)及HRT组(5.96±3.87)(P<0.05)。结论COH组胚胎种植率和临床妊娠率低于HRT组和超排卵组。COH组超生理的雌激素水平可能影响人胚胎种植率和临床妊娠率。     

Methoxychlor accelerates embryo transport through the rat reproductive tract

The estrogenic pesticide methoxychlor (MXC) is known to reduce implantation, and, in our previous work, this reduction has been attributed to a direct effect on uterine function. The present study was designed to investigate the effect of MXC on embryo transport rate, another phenomenon that is vulnerable to estrogenic effects. MXC was administered by gavage, at 0, 100, 200, and 500 mg/kg/day, to groups of rats on Days 1-3 of pregnancy (Day 0 = sperm positive), and the distribution of embryos in the oviducts and uteri of animals was assessed at five time intervals prior to implantation. No effect of MXC was detected by the afternoon of Day 1. On Days 2 and 3 of pregnancy, 200 and 500 mg/kg/day MXC were found to accelerate embryo transport into the uterus; the 500 mg/kg/day dosage also reduced the total number of embryos recovered from the tract. On the third day, 100 mg/kg/day MXC also accelerated embryo transport to the uterus and 200 mg/kg/day MXC reduced total embryo recovery. Until the afternoon of Day 3, most control embryos remained in the oviduct. These data demonstrate that MXC produces a dose-dependent acceleration of embryo transport through the female reproductive tract. When compared with previous work, the current data indicate that such an acceleration is the primary cause of MXC-induced preimplantation embryonic loss when exposure occurs after fertilization.     

Effects of 3H-thymidine on preimplantation mouse embryos in vitro

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.     

来曲唑联合拮抗剂方案在卵巢储备功能低下患者辅助生殖技术中的疗效观察

目的:观察来曲唑联合拮抗剂方案应用于卵巢储备功能低下患者体外受精（in vitro fertilization,IVF）助孕的疗效。方法:将2019年10月至2020年10月期间在东莞市妇幼保健院的波塞冬标准3、4组的卵巢储备功能低下的26例患者应用随机数字表法分为2组。来曲唑组年龄（35.69±4.25）岁,体质量指...     

Increased sister-chromatid exchange frequency in preimplantation mouse embryos after maternal cyclophosphamide treatment before implantation

The effect of a toxic agent in vivo on sister-chromatid exchange (SCE) frequency of preimplantation mouse embryos and bone marrow cells was determined using combined in vivo treatment and in vitro culture in the presence of 5-bromo-2-desoxyuridine (BrdU) for differential staining of the chromatids. In mice exposed to cyclophosphamide (CPA) on day 2 of pregnancy SCE frequency was increased dose-dependently both in embryos and bone marrow cells 1 h after treatment. It returned to control values in bone marrow cells obtained 24 h after exposure but was still significantly increased in the embryos. A closer time-related evaluation of SCE on day 2 of gestation showed a significant increase in SCE in bone marrow cells and in embryos obtained 20-60 min after CPA treatment. Furthermore, SCE frequency was the most sensitive toxicological endpoint to detect embryotoxic effects of CPA treatment before implantation, since it was significantly increased in embryos exposed to 5 mg/kg CPA on day 2 of pregnancy while embryolethality at term and both cytogenetical (structural chromosomal aberrations, micronuclei) and developmental parameters (cell number, differentiation in culture) before implantation did not indicate any toxic effect.     

Effects of advanced maternal age on development of preimplantation mouse embryos obtained from delayed mating

Rodent embryos resulting from delayed mating grow more quickly than those resulting from normal mating. To clarify the effects of maternal age on this enhanced growth and rapid differentiation of mouse embryos obtained from delayed mating, we studied normal (0 h after ovulation) and delayed mating (6 h after ovulation) subgroups in two different age groups (middle-aged, 9-11 months; elderly, 13-15 months), and compared the number of cells and the proportion of blastocyst formed (morphologically advanced blastocysts) among them. In middle-aged female mice, preimplantation embryos derived from delayed mating progressed more rapidly than their normally mated counterparts. The number of cells and the proportion of blastocyst formed among embryos from delayed mating were almost 3 h ahead of those of normal embryos. In the delayed mating subgroup from elderly mice, an enhanced rate of development was not observed. There was no difference in development of normally mated embryos from middle-aged females compared to each counterpart from elderly female mice. Preimplantation mouse embryonic development from delayed mating may be affected by advanced maternal age.     

Reproductive effects of chlorpromazine exposure to female mice: cell proliferation disadvantage revealed by the Chimera Embryo Assay

Administration of chlorpromazine-HCl at 5 to 15 mg/kg bodyweight to pregnant CD-1 mice at 24 h after human chorionic gonadotropin (hCG) (20-23 h after mating) inhibited blastocyst formation and reduced the cell number of embryos recovered at 95 h after hCG. When embryos are recovered at the two- to four-cell stage (48-50 h after hCG) and cultured for an additional 47 h (to 95 h after hCG) or 72 h (to 120 h after hCG), blastocyst formation and embryo cell number were similarly reduced. When the dose range was reduced to 0.5 to 2 mg/kg bodyweight, no significant effect of the drug was observed on blastocyst formation or on embryo cell number. However, when aggregation chimeras were formed between embryos recovered from drug-exposed females and from untreated females, a decrease in cell proliferation rate of the embryo from the drug-exposed female was observed at a dose of 2 mg/kg bodyweight. This result indicates that exposing pregnant mice to chlorpromazine-HCl at doses as low as 2 mg/kg bodyweight can induce a potential for decreased cleavage rate in their pre-implantation embryos that can be revealed by challenging those embryos by direct contact with embryos from nonexposed females. Finally, when four-cell stage embryos recovered from untreated females cultured in the presence of chlorpromazine (0.1-25 mM), blastocyst formation and embryo cell number were significantly reduced in a dose-dependent manner. This last result suggests that in vivo the drug may act directly on the embryo from the pronuclear stage to the early morula stage of development.     

Decreased superovulation in adult mice following neonatal exposures to technical methoxychlor

To examine the effects of technical methoxychlor (MXC) on superovulation, neonatal mice received intraperitoneal (i.p.) injections of either sesame oil, 10 μg of estradiol 17β, or 0.1, 0.5, or 1 mg of technical MXC. At 2 and 4 months, half of the mice received a superovulatory regimen of 10 IU pregnant mare's serum gonadotropin followed by 10 IU human chorionic gonadotropin. The mice were sacrificed 15 to 20 h later, the number of ovulated oocytes were counted, and the ovaries were removed for histology. In the lowest MXC dose, the ovaries appeared normal and at 2 months, ovulated the same number of oocytes as controls. Estradiol or the highest two MXC closes induced ovarian atrophy. Following gonadotropin injections, these ovaries also ovulated oocytes. However, the number of oocytes recovered from experimental mice exhibited a time- and dose-dependent decline, and by 4 months, their number was significantly reduced. Neonatal exposures to MXC reduces ovulatory rates and ovarian functions in adults.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on preimplantation mouse embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant that can exert developmental toxicity. To investigate the stage-specific effects of TCDD on preimplantation embryos, we exposed mouse embryos to TCDD at different stages (1-, 2-, and 8-cell) and collected them at different stages of development (the 1- or 2-, 8-cell, and blastocyst stage, respectively). Semiquantitative RT-PCR revealed increased constitutive gene expression of the arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) at the 1-cell stage, decreased expression at the 2- to 8-cell stage, and increased expression again at the blastocyst stage, and addition of TCDD to media did not affect their mRNA levels. Interestingly, no cytochrome P4501A1 (CYP1A1) mRNA was detected in embryos at the 1-, 2-, and 8-cell stages after exposure to 10 nM TCDD for 12 or 24 h, whereas CYP1A1 mRNA was significantly increased at the blastocyst stage in response to TCDD, and its induction was found to be concentration-dependent on TCDD exposure from 0.01 to 10 nM for 24 h. In addition, no significant differences in development rate of preimplantation embryos, cell number of blastocyst embryos, or apoptotic indices, such as TUNEL-positive cell number or Bax/Bcl-2 expression ratios were observed at the blastocyst stage between TCDD-exposed groups and non-exposed group. These results suggest that the sensitivity to TCDD differs with the embryonic stage, which may reflect an ability of embryos to adapt to environmental stressors, such as dioxins.     

基因重组人卵泡刺激素用于非低反应人群卵泡期长效促排卵方案的疗效分析

目的比较2种基因重组人卵泡刺激素(rFSH)制剂用于卵泡期长效促排卵方案的非低反应人群的疗效及安全性。方法回顾性纳入首次行卵泡期长效促排卵方案体外受精/胞质内单精子显微注射技术(IVF/ICSI)助孕治疗的非卵巢低反应不孕患者,均行控制性卵巢刺激(COS)卵泡期长效促排卵方案治疗,根据使用r FSH不同分为国产rFSH组(267个周期)和进口rFSH组(240个周期)。比较2组患者在超促排卵过程中的卵巢反应、用药情况、卵子受精及胚胎情况、新鲜胚胎移植及胚胎解冻移植后的妊娠率及活产率。结果超促排卵过程中,国产rFSH组HCG注射日雌二醇(E2)水平低于进口rFSH组(P<0.05),2组Gn总量、Gn使用天数、减数分裂中Ⅱ期(MⅡ)卵数、D3优质胚胎数、获得优质囊胚数等差异均无统计学意义(P>0.05)。主要疗效指标中,国产rFSH组及进口rFSH组的平均获卵数、新鲜胚胎移植周期临床妊娠率、冻融移植周期临床妊娠率等差异均无统计学意义(P>0.05),且2组患者的一次COS累积活产率比较差异均无统计学意义(P>0.05)。结论非低反应患者在卵泡期长效促排卵方案中使用...     

Development and sister chromatid exchange of mouse morulae and blastocysts cultured in rat serum containing active metabolites of cyclophosphamide

To establish an in vitro test system in which sera from animals treated with various chemicals can be tested for embryotoxic effects during the preimplantation period, mouse morulae and blastocysts were cultured in the presence of rat serum (RS) from animals which had been treated with cyclophosphamide (CPA). Development during in vitro culture for 96 h, cell number, chromosomal aberrations and sister chromatid exchange (SCE) were the end-points tested in exposed embryos. SCE frequency was the most sensitive parameter, indicating embryotoxic effects in preimplantation mouse embryos after only 1 h of exposure to RS-CPA.     

Effects of repeated maternal oral exposure to low levels of trichlorfon on development and cytogenetic toxicity in 3-day mouse embryos

Trichlorfon is a widely used broad-spectrum agricultural insecticide. Few studies have evaluated the effects of trichlorfon on developing fetuses, especially at early stages of development after low-level maternal exposures. In this study, we evaluated the direct effects of trichlorfon on preimplantation mouse embryos after 30 days of maternal exposure (2, 10 and 50 mg/kg/day) via drinking water.On gestation day 3 (dg3), blastocysts were collected and evaluated for changes in gross morphology; cell number; the presence of interphase, metaphase, micronuclei (MN) cells and fragmented and pycnotic nuclei. Embryos in the 50 mg/kg/day group had a significantly reduced mean cell number per embryo. Furthermore, there was a significant increase in the frequency of pycnotic nuclei and an absence of metaphase cells in the 50 mg/kg/day treated group. None of the developmental endpoints evaluated were observed in the 2 and 10 mg/kg/day trichlorfon-treated groups. A simultaneous decrease in the cell number and an increase in the frequencies of absent metaphases and pycnotic nuclei indicate that embryonic developmental deficits observed in the 50 mg/kg/day exposure group were associated with cytotoxicity.     

Effect of repeated gonadotropin stimulation on ovarian reserves and proliferation of ovarian surface epithelium in mice

This study aimed to evaluate the effect of repeated ovarian stimulation (OS) on the ovarian follicular population and morphology in female mice and its influence on the embryo’s developmental ability, and the profile of the ovarian surface epithelium (OSE). A total of 75 mice were enrolled in this experiment and randomly assigned into three groups: repeated ovarian stimulated group [n = 25; receiving 5 IU pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotropin (hCG) at 6 day intervals for 5 cycles]; single ovarian stimulated group (n = 25; receiving 5 IU PMSG and hCG for 1 cycle), and control group (n = 25; without additional treatment). The follicle number at various stages and the morphologies were recorded respectively in the three groups. The harvested oocytes or embryos, cleavage rate, good quality embryo rate, and blastocyst production rate were counted and calculated, and the proliferations of ovarian surface epithelium were evaluated respectively. In the three groups, the single ovarian stimulation treatment significantly increased the mean number of ovarian oocytes or embryos (39.25±10.77 one-cell embryos/female); on the other hand, repeated gonadotropin stimulation obtained the lowest mean number (5.15± 2.81 eggs/female, P<0.01). Repeated ovarian stimulation also tended to decrease normal follicles of primary follicles (66.67%) and secondary follicles (72.86%), and got the lowest cleavage rate (67.47%), lowest good quality embryo rate (2.41%), and lowest blastocyst production rate (0). The OSE cells adjacent to the antral follicles and corpus luteum (CL) in the repeated ovarian stimulated group (81.8%) had a significantly higher proliferation rate than the other groups. The proliferation rate of the OSE in the single ovarian stimulated group (56.4%) was significantly higher than that in the control group (37.5%) (P<0.01). In conclusion, single ovarian stimulation may produce more oocytes/embryos. However, repeated gonadotropin stimulation may have a negative effect on the ovarian follicular quality, the number of mature retrieved oocytes, and the embryo quality, even increasing the chance of ovarian cancer. All authors contributed equally to this work.     

In vitro culture of preimplantation mouse embryos and day 12 limb-buds: Effects of serum and albumin

The effects of three different protein sources at different concentrations on the growth and development of preimplantation mouse embryos and day 12 mouse limb-buds in culture were studied. Mouse embryos and forelimb-buds were cultured with a range of concentrations (5.5 to 42%) of either donor bovine serum (DBS) or fetal bovine serum (FBS), or (0.2 to 0.8%) bovine serum albumin (BSA). After 48 h in culture, the rate of embryo development was significantly higher in 5.5% DBS than in all other groups (P < 0.05). The embryo hatching rate was higher in 21% FBS, 42% FBS, and all DBS groups than in serum-free medium, and all BSA groups (P < 0.05). Morphologic analysis of cultured limb-buds at 72 h revealed that total, paw, and cartilage area were greater (P < 0.05) in the serum-free medium than in all other groups. Shape factor analysis suggested that 5.5% DBS was most beneficial to mouse limb-bud development. No differences were seen in DNA or protein content of limb-buds among groups. Results suggest that mouse forelimb-buds can be succesfully cultured in serum-free medium and that high concentrations of FBS and DBS may be detrimental for preimplantation embryo and/or limb-bud growth and development.     

Acrylamide causes preimplantation abnormalities in embryos and induces chromatin-adducts in male germ cells of mice.

Acrylamide, a known male postmeiotic germ cell mutagen, caused a dose-dependent increase in the frequency of morphologic abnormalities in preimplantation embryos. Single-cell eggs, growth retardation, and blastomere lysis were detected after paternal treatment with acrylamide (10 to 50 mg/kg, 5 d). The major effects were seen at weeks 1 to 3 after male treatment, with the highest level of abnormalities at the first week (> 90% vs. 5% in control). The frequency of abnormal four-day embryos was similar to preimplantation loss assessed at 15 to 16 d p.c. A > 100-fold elevation of chromatin adducts in sperm was observed during 1st and 2nd week after treatment, after which adduct levels decreased to baseline level. However, morphologic defects in embryos are not fully explained by the spermatid adduct curve. These findings demonstrate the effects of paternal exposure to acrylamide on preimplantation development and indicate a potential risk to the offspring of men exposed to acrylamide.