Blastocidal and contraceptive actions by an extract and compounds from endod (Phytolacca dodecandra)

An extract consisting primarily of the saponins derived from the Endod plant (Phytolacca dodecandra), three purified trisaccharides derived from the extract, and a synthetic disaccharide were tested for antifertility activity on Days 1, 4, and 6 of pregnancy. Known quantities of the crude extract or purified compounds were injected into one of the two uterine horns of rats, and the contralateral horn served as a control. Separate control groups received physiological saline in one of the two horns. Vaginal smears were routinely observed each morning, and the presence of vaginal sperm determined Day 1 of pregnancy. Then, intrauterine injections were performed by surgery on Days 1, 4, and 6 of pregnancy. Treatment with proper dose levels of the crude saponin extract, lemmatoxin, and oleanoglycotoxin-A was found to be capable of preventing pregnancy or reducing the embryonic count on all three days. Lemmatoxin-C-C' was also tested on Day 1 and found effective at proper dose levels. On Days 1 and 4, oleanolic acid cellobioside had little or no effect on pregnancy when injected in utero at doses up to 1,000 μg. Endod extract caused substantially lower decreases in surface tension of water than Nonoxynol-9, yet showed 5 to 10 times the antifertility activity when administered on Day 1. To test whether a substance acted on the uterine horn to prevent implantation, Endod extract was injected on Day 1 into one of two uterine horns at a high level, sufficient to prevent pregnancy on the treated side. When the oviducts were flushed the third day after mating, 4 out of 5 rats had embryos on the treated side which were in similar stages of development as on the untreated side. Under the conditions of these experiments, there was no obvious damage to the uterine endometrium observed histologically due to treatment with purified compounds. It is suggested that this or related classes of compounds may be useful as abortifacients during early stages of pregnancy.     

Flowers of Hibiscus rosa-sinensis, a potential source of contragestative agent. III: Interceptive effect of benzene extract in mouse

In mouse, oral administration of the benzene extract of Hibiscus rosa-sinensis flowers at a dose level of 1 gm/kg body weight/day from day 5-8 of gestation led to termination of pregnancy in about 92% of the animals. The effect was associated with a significant fall in peripheral level of progesterone and increase in uterine acid phosphatase activity, as measured on day 10. The ovary exhibited signs of luteolysis, and the corpus luteal delta 5-3 beta -hydroxysteroid dehydrogenase activity decreased markedly. The interceptive effect of the extract was prevented completely by exogenous progesterone (1 mg/mouse/day) or chorionic gonadotropin (1 I.U./mouse/day) and partially (62.5%) by exogenous prolactin (500 micrograms/mouse/day). In unilaterally pregnant mouse having trauma-induced deciduomata in the sterile horn, the extract caused resorption of the fetuses, and regression of the deciduomata accompanied by reduction in weight of the ovaries. Luteolysis, may be due to interference with the luteotropic influence, and a consequent fall in plasma level of progesterone have been suggested as the plausible cause of termination of pregnancy.     

Antifertility activity of Montanoa tomentosa (zoapatle)

According to folklore medicine, the Mexican plant zoapatle ($\text{Montanoa tomentosa}$) possesses antifertility activity in women. We report here the effect of various isolated preparations from this plant on early pregnancy in several rodent species including the mouse, rat, hamster, and guinea pig. When an aqueous extract of the leaves similar to the tea utilized in folklore medicine was administered orally during early stages of pregnancy, no antifertility activity could be detected. Day 22 pregnant guinea pigs, however, provided an animal model which allowed conservation of test materials and which showed the antifertility activity of the plant extracts. Purer fractions derived from the plant were more potent in this assay system when administered either intraperitoneally or orally. As the purity of the extracts (and hence the quantity of active ingredient administered) increased, we were able to demonstrate inhibition of implantation in rats and mice when administered on days 1–6 and in hamsters when administered on days 4–6 of gestation. Preliminary data indicate the plant extracts are not estrogenic. It is concluded that zoapatle plant extracts possess unique antifertility activity.     

Role of energy metabolism in the pregnancy interceptive action of Ferula assafoetida and Melia azedarach extracts in rat

Ethanolic extract of Ferula assafoetida and chloroform fraction of Melia azedarach, both devoid of estrogenic activity, were examined for their pregnancy interceptive property. Treatment of rats from days 1 to 7 of pregnancy with either of the plant extracts resulted in pregnancy failure in about 65-85% of the animals. The possible role of energy metabolism in the antifertility action was investigated by measuring changes in activities of the key enzymes of carbohydrate metabolism in uterus on day 7 of pregnancy. It was observed that on the day 7 of pregnancy, one key enzyme of glycolytic pathway (phosphofructokinase) was significantly reduced in the uteri of treated rats as compared to controls. Hexosemonophosphate pathway also appeared to be sensitive to treatment with the plant extracts and showed an inhibitory effect on the enzyme activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Oxidative energy metabolism through tricarboxylic acid cycle, which is considered to be the main source of energy to the uterus at this stage, was maximally affected by the treatment with several enzymes showing significant inhibition. The two plant materials appeared to interrupt the latter metabolic pathway more significantly. It is thus concluded that plants lacking phytoestrogens may intercept pregnancy by their ability to disrupt energy metabolism in rat uterus during implantation, especially the oxidative pathway.     

Flowers of Hibiscus rosa-sinensis, a potential source of contragestative agent: II. Possible mode of action with reference to anti-implantation effect of the benzene extract

Benzene extractives of Hibiscus rosa-sinensis flowers, administered during day 1-4 of gestation, exerted anti-implantation effect without affecting the tubal transport of zygote. On day 4, normal number of blastocyst was present in the uterus but they did not implant. However, as studied by pontamine blue reaction, it was evident that hyper-permeability of the endometrial capillaries which is the earliest known response of a receptive endometrium to any kind of deciduogenic stimulus was inhibited by the extract. The magnitude of decidualization, as assessed by weight of the traumatized uterine horn and supported by the histological pictures of the uteri was significantly lower in comparison to that of the controls. Ovarian structure exhibited signs of luteolysis. Inadequate progestational development of the endometrium due to interference with the conditioning of the uterus with progesterone during prenidatory phase of pregnancy has been suggested as the plausible cause of the extract-induced implantation failure.     

Postcoital ingestion of the aqueous extract of Erythrina falcata Benth prevents pregnancy in the mouse

AIM: We examined whether the aqueous extract of Erythrina falcata, reputed to be a contraceptive in Peruvian folklore, could prevent pregnancy in the mouse. METHODS: Female mice on Day 1 of pregnancy were given aqueous extract of E. falcata or tap water (control) orally for 4 days. On Day 4 of pregnancy, animals were killed and the embryos were flushed from oviducts and uterus to examine their developmental stage, cell number, mitotic index and micronuclei frequency. Other mice were killed on Day 12 of pregnancy to determine the number of implantation sites. RESULTS: Ingestion of E. falcata diminished the percentage of embryos that progressed to blastocyst stage, reduced the cell number and mitotic index, and increased the micronuclei frequency of early embryos. The number of implantation sites was also reduced in females treated with E. falcata. CONCLUSION: The aqueous extract of E. falcata, ingested during early pregnancy, disturbs preimplantation embryo development and implantation in the mouse. These results provide the first experimental evidence of the contraceptive properties of the aqueous extract of E. falcata.     

A relationship between estrogenicity and antifertility activity of 1-diphenylmethylenyl-2-methyl-3-ethyl-4-acetoxycyclohexane (ORF 8511) and similar nonsteroidal anti-implantive agents

Several nonsteroidal estrogens, such as ORF 3858 and F6103, which inhibit pregnancy in experimental animals when given postcoitally, have been previously described. ORF 8511 (1-diphenylmethylenyl-2-methyl-3-ethyl-4-acetoxycyclohexane) which is structurally similar to these compounds was studied for its postcoital antifertility activity and estrogenicity in rats, hamsters, mice and rabbits. Results of these studies suggest a relationship between these two biological endpoints. ORF 8511 produced uterotropic stimulation in the rat at microgram doses and totally inhibited implantation at 250 μg/kg/day administered on days 1–6 of pregnancy. However, the other species were considerably less sensitive to the compound with respect to both parameters. The compound stimulated tubal transport in rats at its minimum effective dose for antifertility activity as did diethylstilbestrol, a known estrogen. In the hamster, a species relatively insensitive to the antifertility effect of ORF 8511, endogenous estrogen titres during early pregnancy were higher than those in the rat. These data suggest that ORF 8511 and similar nonsteroidal compounds may owe their postcoital antifertility activity to their estrogenicity and that estrogens may act as pharmacological agents only in species with low normal endogenous estrogen titres.     

Effects of the hydromethanolic extract of Austroplenckia populnea (Celastraceae) on reproductive parameters of male rats

Austroplenckia populnea (Reiss.) Lundell. was selected for this study because it has been shown that some plants from the Celastraceae family have antifertility effects. Twelve adult male rats were treated with hydromethanolic extract made from the leaves, 500 mg/kg/day, orally, for 70 days. Distilled water was administered to the control animals (n = 10). At the end of the experiment, and before killing the rats, their sexual behavior was evaluated. The number of intromissions, latencies to first mount and ejaculation, and first intromission after ejaculation were significantly reduced in the treated group, but the total number of ejaculations did not differ from the control group. The weight and histology of the reproductive organs, sperm production, spermatogenesis, prostate fructose content, cauda epidydimides duct diameter, and sperm morphology were not affected. Sperm concentration in cauda epidydimides was significantly decreased. The results showed that A. populnea has effects on male rat reproduction, affecting the sexual behavior and epididymal sperm concentration.     

Post-coital contraceptive agents from extracts of Sri Lankan alcyonacean soft corals--I.

Dichloromethane-methanol (1:1) extracts of ten species of alcyonacean soft corals were screened for post-coital interceptive effects on female rats, using oral administration, from day 1 to day 7 of pregnancy. Significant suppression of fertility was observed in the animals treated with the crude extracts of Sarcophyton ehrenbergi (250 mg/kg/day, P less than 0.05; 500 mg/kg/day, P less than 0.001), Sinularia crispa (500 mg/kg/day, P less than 0.05; 1000 mg/kg/day, P less than 0.001), Sinularia abrupta (1000 mg/kg/day, P less than 0.001), Sinularia spp--III (500 mg/kg/day, P less than 0.05). This antifertility effect appears to be due to interruption of embryonic events during implantation and/or pre-implantation periods.     

Postcoital antifertility effect of piperine

The antifertility activity of piperine was investigated in pregnant mice when given by various routes of administration and at different periods of gestation. Piperine effectively inhibited implantation, produced abortion and delayed labor when it was given from day 2 through 5, day 8 through 12 and day 15 until labor, respectively. At the same dose level which interrupts pregnancy, piperine did not affect the estrous cycle. Neither uterotropic, antiestrogenic nor antiprogestational property was observed. Additionally, piperine also inhibited uterine contraction both in vivo and in vitro. These results suggested that the antifertility activity of piperine did not operate through any hormonal actions or uterotonic activity.     

Effect of Maytenus ilicifolia Mart. on pregnant mice

Maytenus ilicifolia Mart. is used in Brazilian herbal medicine particularly for stomach disorders, but it is also used, as in other parts of South America, for fertility control. To verify its potential as an abortifacient, the lyophilized hydroalcoholic extract of its leaves was administered orally at a dose of 1000 mg/kg/day to mice between the first and third day of pregnancy (DOP), between the forth and sixth DOP, or between the seventh and ninth DOP. The extract caused a pre-implantation embryonic loss, but it did not have an effect on implantation or organogenesis. Morphological alterations of the reproductive system, not an embryotoxic effect, were not found. Estrogenic activity of the extract, exhibited by an uterotrophic effect, suggests that it may be interfering with the uterine receptivity to the embryo.     

Flowers of Hibiscus rosa-sinensis, a potential source of contragestative agent: I. Effect of benzene extract on implantation of mouse

In mouse, the benzene extract of $\text{Hibiscus rosa-sinensis}$ flowers was administered at four different dose levels (250–1000 mg/kg body weight/day) from day 1–4 postcoitus. Anti-implantation response and associated changes in the uterine chemical composition were studied. With an increase in the dosage of the extract, the percentage of implantation failure increased. At the dose level of 1 gm/kg body weight the extract led to failure of implantation in 93% of the mice. The effect was accompanied by adversely altered uterine weight, its protein content and alkaline and acid phosphatase activity. In another experiment, influence of the extract on uterine uptake of progesterone was studied in bilaterally ovariectomized mice treated with or without estrogen. It exerted neither inhibitory nor stimulatory influence on uterine progesterone uptake in untreated castrated mice but the estrogen-induced increase in the uptake level was significantly inhibited by the extract. Failure of uterine bed preparation due to antiestrogenic potentiality of the extract has been discussed as the plausible cause of implantation failure.     

体外受精-胚胎移植术移植日子宫内膜容受性的超声学研究

目的:探讨体外受精-胚胎移植(IVF-ET)/卵泡浆内单精子显微注射(ICSI)周期胚胎移植日胚胎种植率的影响因素。方法:将实施IVF/ICSI的112个周期根据助孕结局分为妊娠组和非妊娠组,比较两组的E2、P、E2/P及子宫内膜厚度/形态、宫腔深度,并与自然周期模拟移植日子宫内膜厚度/形态、宫腔深度进行自身对照研究。结果:两组超排卵周期胚胎移植日宫腔深度、内膜厚度均显著高于自然周期模拟移植日(P0.001);两组胚胎移植日C型内膜的比率均高于模拟移植日(P0.05),而妊娠组与非妊娠组之间宫腔深度、内膜厚度、C型内膜比率无统计学意义;妊娠组E2、E2/P显著低于非妊娠组(P0.05)。结论:超排卵治疗改变了子宫容积和内膜形态,通过生殖激素的变化而影响IVF/ICSI的妊娠结局。     

Contraceptive and hormonal properties of the stem bark of Dysoxylum binectariferum in rat and docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction

BACKGROUND: This study was aimed to investigate the pregnancy interceptive activity of the stem bark of Dysoxylum binectariferum Hook. f. administered during the pre- and peri-implantation periods and immediately after implantation by oral route in adult female Sprague-Dawley rats. STUDY DESIGN: Ethanolic extract and its fractions were administered to female rats on Days 1-10, Days 1-7, Days 1-5 or Day 1 postcoitum by oral route. At autopsy on Day 12, the number and status of corpora lutea and implantations were recorded. For estrogenic activity, ovariectomized immature rats received the test extract or the vehicle once daily for 3 days and at autopsy on Day 4, uterine weight and status of vaginal opening and extent of vaginal cornification were recorded. For antiestrogenic activity, the extract was administered along with ethinyl estradiol. Docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction, to estrogen receptor (ERalpha) was conducted using AutoDock 3.0.5 on a Linux workstation. RESULTS: The ethanolic extract intercepted pregnancy in rats at a daily dose of 500 mg/kg on Days 1-7 postcoitum. On fractionation, the activity was localized in the chloroform fraction, which inhibited pregnancy in all females at the 35-mg/kg dose on Days 1-7, at the 50-mg/kg dose on Days 1-5 or at the single 300-mg/kg dose on Day 1 postcoitum. Chromatography of this fraction yielded an alkaloid, rohitukine, which prevented pregnancy at the 10-mg/kg dose administered on Days 1-7 but was partially (45%) effective at this dose when administered during the entire preimplantation period and ineffective even at 10 times this dose when administered only on Day 1 postcoitum, except that there was a significant reduction in implantation number in pregnant females. While the active chloroform soluble fraction was devoid of any estrogen agonistic or antagonistic properties, a mild uterotropic effect without induction of premature opening of vagina or cornification of vaginal epithelium was observed in rohitukine at the 10-mg/kg dose. Rohitukine, with an almost similar molecular size (mol. wt. 305) as 17beta-estradiol, fits ideally into the hydrophobic pocket of ER. While it does not appear to simultaneously interact with GLU353, ARG394 and HIS524 as estradiol to elicit frank estrogenic response, different conformations of the ligand or its metabolite(s) might acquire geometry with phenolic groups at C-3', C-5 and C-7 positions disposed in a fashion to interact with active site(s) of ER, which might be responsible for its contraceptive and/or weak uterotropic effects. The absence of a basic side chain directed toward the antiestrogen binding site (ASP351) on the receptor appears to be responsible for the lack of any estrogen antagonistic activity. CONCLUSIONS: Findings demonstrate the antifertility activity of the ethanolic extract of D. binectariferum, its chloroform soluble fraction and rohitukine. Efforts are being made to enhance the anti-implantation activity of rohitukine by structural modifications.     

预备周期子宫内膜glycodelin、galectin-3表达评估IVF子宫内膜容受性

目的:探讨子宫内膜着床窗口期glycodelin、galectin-3表达评价体外受精-胚胎移植(IVF-ET、简称IVF)子宫内膜容受性。方法:接受IVF治疗患者,前一月经周期HCG日,阴道超声子宫内膜厚度和形态;着床窗口期子宫内膜采用RT-PCR技术检测glycodelin mRNA、galectin-3mRNA表达,评价其与妊娠的关系。结果:185例IVF患者,90例妊娠,95例未妊娠,比较妊娠组HCG日内膜厚度和三线型比例与IVF未妊娠组无统计学差异,妊娠组子宫内膜着床窗口期glycodelin、ga-lectin-3的表达明显高于未妊娠组。结论:子宫内膜着床窗口期glycodelin、galectin-3的表达可用于评估子宫内膜容受性;子宫内膜厚度和形态不能完全评价子宫内膜容受性。     

Effects of postcoital insertion of an intrauterine foreign body in rats

The effects of brief exposure to an intrauterine foreign body (IUFB) were studied in pregnant and pseudopregnant rats. Insertion of an IUFB on the morning of day 1 or 2 of pregnancy (day 1 = day of vaginal sperms) and its removal about twenty-four hours later did not influence implantation or decidual growth. However, exposure during days 3–4 inhibited both implantation and deciduomal change. Exposure of upper (ovarian) or lower (cervical) half of the uterus during days 3–4 or the sham procedure of IUFB insertion itself did not prevent implantation but it caused high embryo mortality. Exposure during days 4–5 did not prevent decidual change but it inhibited both implantation and decidual growth. Other studies indicated that exposure during days 3–4 caused: (1) a significant increase in the number of leucocytes in the uterine lumen; (2) stimulated mitosis in the luminal epithelium; (3) advanced from pseudopregnancy day 5 to day 4 the peak in uterine weight and nuclear DNA-dependent RNA polymerase activity; and (4) significantly inhibited uterine weight, RNA and DNA-dependent RNA polymerase response to a decidualizing stimulus. These studies demonstrate that an IUFB can act as an effective postconceptional type of contraceptive in rats. Furthermore, it is suggested that the antifertility effect of an IUFB insertion on day 3 may be mainly due to partial inhibition of uterine responses to ovarian hormones and that of an insertion on day 4 may be mainly due to an inhibitory influence of decidual change on implantation.     

Effects of castor bean extract and ricin A-chain on ovulation and implantation in rabbits

The anti-implantation and antiovulation effects of castor bean extract (CBE) and ricin A-chain (RAC) were evaluated in rabbits. Both CBE and RAC, administered intraperitoneally on days 5–9 of pregnancy, exhibited a pronounced decrease in maternal body weight gain and in death of all fetuses. A significant (p <0.01) decrease of implantation sites resulted after rabbits were treated with RAC on the first 6 consecutive days of pregnancy. When female rabbits were treated with RAC for 10 consecutive days followed by human chorionic gonadotropin (hCG) (50 IU/kg intravenously), there was a 30% reduction in the number of corpora lutae. These data clearly indicate that CBE and RAC possess potent effects on implantation and ovulation in rabbits. The protein contents of castor bean extract, separated by polyacrylamide gel electrophoresis, revealed the presence of several protein bands, ricin toxin being a major constituent of the extract.     

Anordiol produces pregnancy loss in the rat--via the embryo or via the uterus?

Anordiol, the dihydroxylated metabolite of anordrin, is an antiestrogen with estrogenic activity that is known to inhibit fertility. The following study was conducted to determine the mechanism of this antifertility effect. Anordiol was administered orally to rats, prior to implantation, on Day 2 of pregnancy. Control animals were treated with the vehicle only. The effectiveness of the agent in terminating pregnancy was determined on Day 14 of pregnancy. Anordiol was 100% effective in abolishing pregnancy at a dose of 0.6 mg/Kg. Administration of smaller doses resulted in a decreased number of implanting embryos, in a dose-dependent manner. An additional dose of anordiol on Day 3 of pregnancy yielded similar results. To determine whether pregnancy impairment by anordiol is exerted via the embryo or via the uterus, reciprocal embryo transfers were performed. Day 5 blastocysts were transferred into the uteri of pseudopregnant rats. In one set of experiments, the donor rats were treated with anordiol, and in the second set the recipient rats were treated. The results indicate that the effects of anordiol administration are exerted via the embryo as well as the uterus.     

Evaluation of Early Embryonic Loss Induced by Tributyltin Chloride in Rats: Phase- and Dose-Dependent Antifertility Effects

In our previous study, tributyltin chloride (TBTCl) on days 0–7 of pregnancy was found to produce implantation failure in rats. The objective of the present study was to determine the susceptible period for the antifertility effect of TBTCl in rats. Inseminated females were orally administered TBTCl at 8.1, 16.3, or 32.5 mg/kg on days 0–3 of pregnancy, or at 8.1, 16.3, 32.5, or 65.1 mg/kg on days 4–7 of pregnancy. Pregnancy outcome was determined on day 20 of pregnancy. Dosing with TBTCl on days 0–3 of pregnancy at 16.3 mg/kg and higher produced a significant increase in the rate of implantation failure. Dosing with TBTCl on days 4–7 of pregnancy caused a significant increase in the incidence of postimplantation loss at 16.3 mg/kg and higher in females with implantations. No increase in the incidence of fetal malformations was found in any TBTCl-treated groups. It could be concluded that the susceptibility to and manifestation of the antifertility effects of TBTCl vary with the gestational stage at the time of administration. Received: 9 April 1997/Accepted: 12 June 1997     

Prototype for a new class of antifertility agents, 3,5-Bis (dimethylahino)-1,2,4,-dithiazolium chloride

3,5-Bis(dimethylarmino)-1,2,4-dithiazolium chloride [ORF 5513] is considered to be a prototype for a new class of female antifertility agents. When orally administered to rats at dose levels between 0.01 and 0.1 mg/kg/day for 3 days prior to expected ovulation, treatment resulted in a dose-dependent inhibition of ovulation. However, when the compound was administered to animals for 2 days prior to ovulation, ORF 5513 had little or no effect on ovulation at doses as high as 10 mg/kg/day. ORF 5513 also interrupted pregnancy at several stages of gestation, including implantation, placentation and late pregnancy. The minimum effective dose (MED) in the rat for inhibiting implantation (Days 1–6) and interrupting pregnancy immediately after implantation (Days 7–10) appears to be ≤ 10 mg/kg. The MED varies between ≤ 5 mg/kg for days 10–13 or 13–16 to ≤ 20 mg/kg for days 16–19 of gestation. Endocrine bioassays and $\text{in vitro}$ studies revealed that ORF 5513 lacks hormonal activity. Histological studies indicated that the corpora lutea in all animals appeared uninvolved. When the treatment was initiated on day 16 of gestation, the earliest detectable cellular changes occurred in the chorionic villi and chorionic fetal vessels which effectively interfere with fetal circulation. Degeneration of the fetus occurred by day 19 of gestation while maternal placental circulation remained intact.