Ovarian stimulation leads to a severe implantation defect in mice.

The aim of the present study was to evaluate whether ovarian stimulation could induce embryo implantation dysfunction in mice and to explore the possible mechanisms involved. Ovarian stimulation was performed with intraperitoneal injections of 0, 2.5, 5.0, 7.5 and 10.0 IU pregnant mare serum gonadotrophin followed by the same dose of human chorionic gonadotrophin 48h later. A dose-dependent implantation defect in stimulated mice was demonstrated, which can be mainly explained by premature luteolysis and secondary endometrial changes induced by an imbalance in oestradiol and progesterone.

Ovarian reaction and estrus manifestation in delayed puberty gilts after treatment with equine chorionic gonadotropin.

BACKGROUND: Prolonged pre-insemination anestrus (i.e. delayed puberty) is a major contributing factor for culling up to 30% of the replacement gilts at large breeding farm units in Vojvodina. It is imperative to determine if these gilts are acyclic (prepubertal) or cyclic, but just fail to exhibit behavioural estrus. Recent investigations demonstrate that treatment with equine chorionic gonadotropin (eCG) can increase the diestrous phase duration in sexually mature gilts. Based on these finding, the aim of the present studies was to determine the reproductive status of delayed puberty gilts following injection with eCG. METHODS: Two experiments were conducted on a swine breeding farm in Vojvodina. In Exp. 1, 20 prepubertal (acyclic) gilts, and 120 sexually mature (cyclic) gilts were injected with a single injection of 400 IU eCG + 200 IU human chorionic gonadotropin (hCG) or with 1000 IU eCG (cyclic gilts), at d5, d11 or d17 after spontaneous estrus detection, to determine their ovarian reaction and induced estrus manifestation. In Exp. 2, sixty delayed puberty gilts (estrus not detected until 8 month of age, av. 258 days) were culled from breeding herd and slaughtered to determine their reproductive status based on ovarian anatomical features. The second group of gilts (n = 60) was treated with a single 1000 IU eCG injection to determine their reproductive status, based on the interval between eCG injection to estrus detection and duration. The data were analyzed by descriptive statistics, t-test, analysis of variance and Duncan's test in the software package Statistics 10th. RESULTS: Ovulations were induced in 90% of acyclic (sexually immature) and, on average, 93.3% of cyclic (sexually mature) gilts after the eCG injection. On average, 4 days after the eCG injection, estrus was detected in 85% of the treated acyclic (sexually immature) gilts and in 95% (19/20) of the cyclic (sexually mature) gilts, treated with eCG on day 17 after spontaneous estrus detection. The interval from eCG to induced estrus detection was prolonged (av. 25 days) in 95% (19/20) of the sexually mature gilts treated with eCG on day 5 and in 90% (18/20) of gilts treated on day 11 after spontaneous estrus detection (Exp. 1). Forty anestrous gilts reached cyclic pubertal ovarian activity. Estrus manifestation was detected in 56 gilts (93.3% of the total 60 treated prolonged anestrous gilts, av. 259 days of age), after a single 1000 IU eCG injection. Thirty-four gilts (60.7% of the total gilts in estrus) with prolonged eCG to estrus interval (av. 24.7 days) were considered spontaneously cyclic (sexually mature), but behaviourally anestrous before treatment. The remaining 22 (39.3% of the total gilts in estrus) were considered truly sexually immature (acyclic) before the treatment or were eCG injected in the late luteal or proestrous phase of spontaneous estrous cycle (Exp. 2). CONCLUSIONS: In 66.7% of the delayed puberty gilts, pre-ovulatory follicles (PoF), corpora hemorrhagica (CH), corpora lutea (CL), or corpora albicantia (CA) were found on the ovaries upon post mortem examination. These gilts were considered as sexually mature before slaughtering. In 60.7% of the delayed puberty gilts, behavioural estrus was detected an average of 24.7 days following eCG injections. These gilts were considered as eCG treated during the luteal phase (diestrus) of the spontaneous estrus cycle. Both findings suggest that delayed puberty gilts actually reached cyclic pubertal ovarian activity (sexual maturity) before culling from the breeding herd.

Follicular development in pregnant cows after the administration of equine chorionic gonadotropin (eCG): a new insight.

The follicular development in the cow occurs in a wave-like pattern, and it takes place also during pregnancy. In the cow, Equine Chorionic Gonadotropin (eCG) is used for superovulation, but a decrease in total fertility has been reported, likely because of its immunogenic properties in species other than equine. In this regard, immune response has been implicated in follicular growth, ovulation, and placental development. So, aims of our study are to test the safety of eCG administered during pregnancy and characterize the ovarian activity, the quality of oocytes, the hormonal status, and interleukin levels in eCG-treated pregnant cows.

Transcripts of neurokinin B and neurokinin 3 receptor in superovulated rat ovaries and increased number of corpora lutea as a non-specific effect of intraperitoneal agonist application.

Neurokinin B (NKB), a member of the tachykinin family, and its neurokinin 3 receptor (NK3-R) are preferentially found in the central nervous system. Others have recently reported on mRNA from this ligand-receptor system in the uterus and on NK3-R expression increasing with age. NKB and NK3-R mRNAs have also been noted in cumulus cells and oocytes from superovulated rats. Intact ovaries before and after puberty have not been studied. In this study, we stimulated 29-day-old rats by s.c. injections with gonadotropins for estrous cycle synchronization in order to elucidate the NKB-NK3-R system's expression and function in the ovary. Simultaneously, NaCl, the NK3-R agonist (Pro(7))-NKB, the antagonist SB 218795, or thiorphan, a neutral endopeptidase inhibitor of tachykinin degradation, were injected intraperitoneally (i.p.) for 3 1/2 consecutive days. First, we demonstrated NKB and NK3-R transcripts in one rat ovary by RT-PCR. No significant mRNA differences were noted between immature ovaries and superovulated ovaries in any of the i.p. applications. Second, the possible role of NK3-R on the ovulatory process was verified by counting corpora lutea (CL) and CL cysts in serial sections of the other ovary derived from the four different groups and embedded in paraffin wax. CL and CL cysts were noted in greater numbers in the pharmacologically treated groups than in the saline-treated group. To validate possible drug effects on the peritoneum, we additionally studied pieces of the omentum majus and retroperitoneal fat tissue. Both tissues were heavily infiltrated by granulocytes similar to a non-specific inflammatory response. The saline-treated group as well as the pharmacologically treated groups appeared to develop this unexpected side effect to a similar degree. We conclude that transcripts of NKB and NK3-R are present before and after puberty in the rat ovary and appear to be expressed at similar levels which may indicate a role for the NKB-NK3-R system in follicle growth. The effect of increased CL formation after application of the NK3-R agonist i.p. is related to a non-specific response.

Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice.

Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.

[Malformations and abnormality of the sex ration after hormone treatment in mice (author's transl)]. / Missbildungen und Störung des Geschlechtsverhältnisses nach Hormonbehandlung bei Mäusen

Ovulation was induced in 90 randomly-bred virgin mice (Swiss albino) by the i.p. injection of 5 I.U. pregant mare's serum and human chorionic gonadotropin, respectively, with an interval of 48 hours between injections. Pregnancy occurred in 32 animals following normal fertilization. Caesarian section was generally performed on account of blocked delivery and the progeny placed with foster mothers. A statistically-significant shift in the sex ration (to the disadvantage of the males) was observed. The incidence of malformations of the extremities was significantly higher than in a control group. In comparison with control embryos which were also born by Caesarian section, the chance of survival of the progeny of hormone-injected females appeared distinctly reduced. A possible correlation between hormone-induced ovulation and abnormalities in the progeny is discussed in this paper.

Administration of a gonadotropin-releasing hormone agonist affects corpus luteum vascular stability and development and induces luteal apoptosis in a rat model of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotropins followed by the administration of human chorionic gonadotropin (hCG) to trigger the final steps of oocyte maturation. Gonadotropin-releasing hormone (GnRH) analogs are thought to be effective in preventing this complication and a clinical trial has found a lower incidence of OHSS in patients treated with these molecules. Our aim was to analyze the in vivo effect of a GnRH-I agonist on corpus luteum development and regression, ANGPT-1, ANGPT-2 and Tie-2 protein expression and luteal blood vessel stabilization, the expression of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) and cell proliferation, in ovaries from an OHSS rat model. To this end immature female Sprague-Dawley rats were hyperstimulated and treated with a GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) administration until the day of hCG injection for 5 consecutive days. Blood and tissue samples were collected 48h after hCG injection. Vascular endothelial growth factor VEGF levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxylin and eosin (H&E) stained slides. Luteal blood vessel stability, cell proliferation and apoptosis were assessed by immunohistochemistry for SMCA, PCNA, and TUNEL, respectively. P450scc, StAR, FLK-1, ANGPT-1, ANGPT-2, Tie-2 and PCNA protein levels were evaluated by Western blot from dissected corpora lutea (CL). The treatment with the GnRH-I agonist significantly decreased serum progesterone and estradiol levels as well as P450scc and StAR protein expression in the untreated OHSS group. In addition, the agonist significantly decreased the number of CL in the OHSS group, as compared with the untreated OHSS group. In the OHSS group, the area of periendothelial cells in the CL was larger than that of the control group. However, the treatment with the GnRH-I agonist significantly reduced the area of periendothelial cells in the CL in the OHSS group. The luteal levels of ANGPT-1 and its receptor Tie-2 significantly increased in the OHSS group when compared with the control group. Conversely, the administration of the GnRH-I agonist significantly decreased the levels of these factors in the CL from the OHSS group, as compared with the untreated OHSS group. In addition, the treatment with the GnRH-I agonist reduced the diameter of CL and decreased CL cell proliferation as compared with that observed in the untreated OHSS group. Finally, the GnRH-I agonist increased apoptosis in the CL from the OHSS group. In conclusion, these results show that GnRH-I agonist exerts diverse actions on the CL from a rat OHSS model. The decrease in P450scc, StAR, ANGPT-1 and Tie-2 expression, blood vessel stability and luteal proliferation leads to CL regression in the ovaries from OHSS rats. Moreover, our results suggest that the downregulation of ANGPT-1 and its receptor is a possible mechanism whereby GnRH-I agonists could prevent early OHSS.

FSH is superior to eCG for promoting ovarian response in Chinese Bamei gilts.

The Bamei gilt is a Chinese native breed located in northwest China, which adapts to the extremely dry and cold environment and is distinguished for its excellent reproductive and maternal characters. To ensure sufficient numbers of embryos for transgenic and nuclear transfer research, hormonal induction of gilt estrus and superovulation may be necessary. The objective of this study was to compare the superovulation effects of equine chorionic gonadotropin (eCG, Group A) and FSH (Groups B-D) in Chinese Bamei gilts. The results show that though eCG could produce more corpora lutea (CL, 14.3) than the control (CL, 9.2), and the FSH treatments had significantly increased the number of CL compared with the eCG treatment. Within the different FSH protocols, the numbers of CL were significantly greater in Groups B (CL, 77.8) and C (CL, 66.8) than in Group D (CL, 42.7), however, ovarian cysts were observed in Groups B and C, but not in Group D. These data suggest that Group D (280 IU FSH) is a suitable protocol to facilitate the development of ovarian follicles and increase the number of useful embryos per gilt for embryos recovery. The optimal FSH protocol of superovulation in Bamei gilts appears to be: D13/100 IU, D14/80 IU, D15/60 IU, D16/40 IU plus prostaglandin (PG) 0.2mg, D17/hCG 1000 IU.

[The appearance of ovarian cysts in young sows after treatment with gonadotropin preparations for estrus induction]. / Untersuchungen über das Auftreten von Ovarialzysten bei Jungsauen nach Behandlung mit Gonadotropinpräparaten zur Zyklusinduktion.

In 72 (46%) of 155 gilts discarded for genetic reasons after performance testing and housed under fattening conditions no heat could have been detected during the first 30 days. The gilts were assigned alternatingly to a control group and four different treatments of delayed puberty. The induction of puberty was carried out by injections of 1000 iu PMSG, 400 iu HCG and 2 mg oestradiol benzoate, 400 iu PMSG and 200 iu HCG and 800 iu PMSG and 400 iu HCG. If there was no estrus gilts were slaughtered 12 days later for examination of the ovaries. Those coming into estrus were slaughtered 8 days after disappearance of estrus. Estrus could be induced in 69 to 94% of the gilts, whereas 40% of the untreated showed estrus signs. After treatment with PMSG and HCG in 40 and 87% of the gilts cysts were found whereas none of the untreated and 26 and 29% of those treated with PMSG und HCG + oestradiol benzoate revealed ovarian cysts. In addition, those gilts that had come into estrus during the first 30 days were given injections of either 1000 iu PMSG or 800 iu PMSG and 400 iu HCG. The injections were made either on the 5th, 10th or 15th day of cycle. In both latter groups significantly more gilts showed standing heat than after treatment at cycle day 5. The results of inspection of the ovaries at slaughter and steroid hormones could not be assigned to a defined stage of the physiological ovarian cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential teratogenicity of gonadotropin treatment for ovulation induction in the mouse offspring.

The teratogenic effects of induced ovulation were studied in mice by using three different doses of pregnant mare's serum (PMS)/human chorionic gonadotropin (HCG) (2.5, 5, or 10 IU) at two different stages of the estrous cycle. The PMS/HCG treatment induced high incidences of external congenital anomalies in the offspring in a dose-dependent manner. This was especially so when the treatment was "out of phase" to the naturally occurring ovulation schedule. The predominant malformations were open eyelids and cleft palate. The problems of extrapolating these findings to humans are discussed.

Gonadotrophin stimulation of donor females decreases post-implantation viability of cultured one-cell hamster embryos.

Hamster one-cell embryos were collected from two groups of donors: females that were super-stimulated with pregnant mare's serum gonadotrophin (PMSG) and females that were not. Embryos were cultured for 72 h and scored for development. Morulae and blastocysts from the PMSG- and non-stimulated females were transferred into contralateral uterine horns of non-stimulated recipient females, so that experimental embryos (from PMSG-stimulated females) and control embryos (from non-stimulated females) were paired within a single recipient. Right and left uterine horns of recipient females were examined 11 days later for the number of implantation sites and fetuses. After 72 h of culture, development to morulae and blastocysts was not significantly different for embryos from PMSG- and non-stimulated females. However, embryos from PMSG-stimulated females compared to controls had significantly reduced mean cell numbers (18 versus 21; P=0.003) and a two-fold decrease in viability post-transfer (20 versus 45%; P=0.02). These findings indicate that gonadotrophin stimulation compromises subsequent developmental competence either during oocyte maturation or in the very early embryo, but it is unclear whether reduced viability is a direct effect or is an indirect consequence of PMSG stimulation.

Four years of induction/synchronization of estrus in dairy goats: effect on the evolution of eCG binding rate in relation with the parameters of reproduction.

Ninety-eight Alpine goats of two herds were followed over 4 years in a program of annual artificial insemination after estrus induction/synchronization, including progestagen administration (vaginal sponge) followed by prostaglandin analog and equine chorionic gonadotrophin (eCG) 48 h before sponge removal. Goats were sampled every 4 hours from the 16th to the 56th following sponge removal, for determination of LH surge and tested for estrus by the presence of a buck. Seven days after AI, endoscopic examination of the ovaries was performed to determine the number of corpus lutea. Pregnancy diagnosis was performed at day 21-22 post AI by determination of plasma progesterone and at day 40-45 by ultrasonography. Parturition, number and sex of kids were recorded. All the goats were sampled before and after each treatment, for anti-eCG antibodies screening. Statistical analysis of the results clearly established a significant effect of the treatments on anti-eCG antibodies. Time of estrus and LH surge were significantly different between herd. The antibodies significantly delayed the time of coming out of estrus as well as the time of LH surge. Two antagonistic effects were evidenced: first, the delayed of time of estrus and time of LH surge in relation with the immune reaction to eCG; secondly, the ahead of time of estrus and time of LH surge during the years of treatment, identical to both herd. The antibodies negatively influenced the percentage of ovulating females as well as kidding rate. Finally, no effect of antibodies on prolificacy was found.

The impact of ovarian stimulation on implantation and fetal development in mice.

The objective of this study was to evaluate, using an embryo donation model, whether impaired oocyte/embryo developmental competence and/or changes in uterine milieu are responsible for the previously observed adverse effects of superovulation with gonadotrophins on implantation and fetal development in mice. Embryos from superovulated and non-stimulated females were transferred to separate uterine horns within the same superovulated or non-stimulated pseudopregnant recipient mice. Embryo development was impaired as a significantly higher proportion of normal embryos from control donors (61%) were blastocysts on transfer day compared with superovulated donors (41%; P = 0.001). The implantation rate in control recipients was significantly reduced after transfer of embryos from superovulated donors (12%) compared with control donors (25%; P = 0.001). Uterine receptivity was impaired in superovulated recipients. The implantation rate of control embryos was significantly higher in control (25%) than in superovulated recipients (7%; P = 0.001). Transfer of embryos recovered from superovulated donors resulted in significantly higher post-implantation fetal mortality in superovulated recipients (69%) than in control recipients (36%; P = 0.01), and the mean weight of live fetuses was significantly lower for fetuses obtained from superovulated recipients (0.51 g) compared with that of fetuses obtained from control recipients (0.72 g; P = 0.006). Hence, ovarian stimulation appears to impair oocyte/embryo quality as well as uterine milieu.

Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice.

The effect of gonadotrophins on pre- and postimplantation development in mice was investigated by superovulating C57BL/6J/Bom females with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) or by inducing ovulation with hCG. In both hormone treated groups, the proportion of abnormal preimplantation embryos increased compared with naturally ovulating animals. Postimplantation mortality increased and the mean number of live fetuses per pregnant mouse decreased in superovulated and hCG-treated mice compared with controls. Embryonic growth was highly retarded. Mean weight of live fetuses in superovulated and hCG-treated mice was reduced and skeletal examination revealed developmental retardation. In conclusion, superovulation as well as induction of ovulation adversely affected embryonic and fetal development.

Treatment with gonadotropins impaired implantation and fetal development in mice.

PURPOSE: The effect of gonadotropins on implantation and fetal development in mice was investigated by superovulation with pregnant mare serum gonadotropin and human chorionic gonadotropin. In a previous study fetal growth was found to be highly retarded. RESULTS: Assessment of implantation in vivo revealed that late implantation did occur. Gestational length was highly extended, the mean number of live fetuses per pregnant mouse was lower and their mean weight significantly reduced. In vitro experiments revealed no significant difference in the rate of blastocyst adhesion and trophoblast outgrowth development. Immunohistochemical staining, however, showed that blastocysts from superovulated mice had smaller trophoblastic outgrowths than control embryos. Staging embryonic development at the time of flushing, however, revealed retarded embryo development in vivo in hormone-treated mice. After correlation with embryonic stage at the beginning of the culture, there was no difference in the size of trophoblastic outgrowths. CONCLUSION: Treatment with gonadotropins impaired implantation and embryonic/fetal development. Changes in maternal milieu, rather than in embryo quality, may be responsible for the adverse effects observed.

Congenital malformations in mice after gonadotropin-induced ovulation.

Virgin mice were treated with gonadotropic hormones in order to induce superovulation; at term the embryos were removed by cesarean section. This treatment induced malformations (mainly forelimb defects and to a smaller extent central nervous system anomalies) as well as an altered sex ratio in the offspring. Both phenomena were statistically significant. These hormone-induced effects on the progeny were significantly dependent on both the dosage of hormones and the time (season) of administration. Because the time of administration (before mating) of gonadotropic hormones does not coincide with the critical period during embryogenic development for the teratogenic induction of malformations in the limbs and in the central nervous system (ca. 8th-12th day of gestation), the investigated defects are interpreted as of mutagenic origin.

Gonadotropin induced reproductive cyclicity in the domestic cat.

Adult queens were treated with one of two sources of follicle stimulating gonodotropin (follicle stimulating hormone or pregnant mares' serum gonodotropin) to induce ovarian follicle development and estrus. Treatment involving single injections of follicle stimulating hormone stimulated mature follicle development but resulted in inconsistent display of sexual receptivity and variable ovulation rates. Single or multiple injections of pregnant mares' serum gonadotropin induced follicel growth, but follicle rupture often occurred prior to estrus and the mating stimulus. When compared to results obtained from queens mated while in natural estrus, cats receiving a series of injections of follicle stimulating hormone or pregnant mares' serum gonadotropin demonstrated ovarian hypersensitivity and produced follicular cysts which failed to rupture. The most effective hormone regimen for inducing follicle growth and estrus consisted of 2.0 mg follicle stimulating hormone injected daily intramuscularly until estrus. Queens receiving this treatment were capable of ovulating following mating or human chorionic gonodotropin administration and were capable of conceiving if mated.