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Embryo transfer in the cat during the non-breeding season.

Studies were conducted to investigate the possibility of embryo transfer in the cat during the non-breeding season. Estrus was induced in 19/22 (86.4%) cats using a porcine pituitary gland preparation. Uterine horns were flushed in 5 cats 6-8 days after mating with expanded blastocysts being collected from 4 cats. One to nineteen blastocysts per cat were transferred to the uterine horns of 6 recipient cats in which ovulation had been induced with HCG. The time differences between time of ovulation in donor and recipient animals were 0.5 days earlier in the recipient (2 cats), 1 day later in the recipient (3 cats), and no difference (1 cat); conception occurred in all the recipients. The ratio of fetuses to transplanted embryos were 1/1, 1/2, 2/3, 2/6, 4/7, and 2/19, respectively. Fetal death occurred in 2 cats at days 22 and 25 and abortion occurred in 3 cats at days 34, 35 and 39. There was a delay in the expulsion of placentae in the animals that experienced fetal death on days 22 and 25, expulsion occurring on days 36 and 56, respectively. One cat was treated with progesterone and carried 2 fetuses to day 66; pregnancy was terminated by cesarean section. In conclusion, it was demonstrated that embryo transfer can be performed in cats in which estrus and ovulation have been induced with porcine pituitary gland preparation during the non-breeding season. However, luteal activity needs to be supplemented by exogenous progesterone administration to maintain pregnancy.

Animals↗

A modified technique of human in vitro fertilization and embryo transfer.

Forty-seven ovulatory women were treated with a modified technique of human in vitro fertilization and embryo transfer. All patients received clomiphene citrate, 150 mg per day, and follicle development was monitored by real-time ovarian ultrasound and serum estradiol measurement. In the 45 patients who underwent laparoscopy, eight patients were found to have a poor response to clomiphene citrate and were dropped from the treatment cycle. Four additional patients had an endogenous luteinizing hormone increase and did not undergo laparoscopy. Nine patients were found to have inaccessible ovaries at the time of laparoscopy. In patients with accessible ovaries, oocytes were recovered in all cases. Embryo transfers were performed in 72% of patients, and 19% of these transfers resulted in pregnancy. This technique of ovulation monitoring appears to increase oocyte recovery and fertilization rate and subsequent implantation success.

Adult↗

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs.

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.

Animals↗

A history of farm animal embryo transfer and some associated techniques.

Events over the last 125 years that have been particularly important to the development of embryo transfer in farm animals are reviewed, arguing that an appreciation of the history of a discipline helps shape its future. Special attention is paid to how the motivations of the scientists involved have changed over time, and how these changes have influenced the practical application of embryo transfer to animal breeding.

Animals↗

The development of an efficient ambulatory in vitro fertilization (IVF) and embryo transfer (ET) program using ultrasonically guided oocyte retrieval.

Until recently most of the current in vitro fertilization and embryo transfer (IVF-ET) programs used laparoscopy for oocyte retrieval and included hospitalization in the treatment. The establishment of a successful ambulatory IVF program that uses ultrasonically guided oocyte aspiration, is described. Between August 1985 and February 1987, 384 percutaneous transvesical ultrasonically and transvaginally guided follicle aspirations were performed on 414 patients, following ovarian stimulation with hMG and hCG. Ninety two clinical pregnancies were confirmed by ultrasound. The clinical pregnancy rate was 26.8% when based on the number of embryo transfers. There were 16 multiple pregnancies (17.4%), 18 miscarriages (19.5%), and two tubal pregnancies (2.1%). The most important factors contributing toward the success of this program were rigid adherence to clinical and laboratory protocols, and the maintenance of a strict quality control. The ambulatory management was very well accepted by the patients, who during the entire period of ovarian stimulation had minimal disruption of their routine activities.

Adult↗

In vitro fertilization in domestic and non-domestic cats including sequences of early nuclear events, development in vitro, cryopreservation and successful intra- and interspecies embryo transfer.

The domestic cat may be used as a model for developing assisted reproduction techniques including in vitro fertilization (IVF), embryo culture, cryopreservation and embryo transfer (ET) for application to threatened and endangered species of non-domestic cats. Interoestrous domestic cats were injected with a total of 1.0-6.0 mg follicle-stimulating hormone (FSH) daily for 4 days and with 100 iu human chorionic gonadotrophin (hCG) on day 5. Follicular oocytes recovered at 26 +/- 1 h after hCG were co-incubated for 4-6 h at 38 degrees C in 5% CO2 with spermatozoa (1-2 x 10(5) motile spermatozoa ml-1) collected by artificial vagina. To determine the timing of sperm penetration and early fertilization events in vitro, oocytes were fixed and examined at intervals from 0.5 to 10 h after sperm exposure. The penetration rate of metaphase II (MII) oocytes at 0.5-3 h was equivalent to that at 3-6 h (95 versus 96%). Second polar body extrusion, pronuclear formation and apposition were observed at 2, 6-8 and 10 h, respectively. Simple (Tyrode's) and complex (F-10, M-199 and CMRL-1066) culture media with 10% fetal calf serum were compared for their ability to support development to the morula or blastocyst stage during culture periods of 96-168 h after IVF. The average number of cells per embryo was similar (P > 0.05) in the various media at each interval except that CMRL-1066 reduced (P < 0.05) development at 96 h if it was used before the two-cell stage. In F-10, neither the presence of intact cumulus cells nor changing to fresh F-10 medium at 48 h affected development at 96 h. Blastocyst development at 168 h was similar in both F-10 (18%) and Tyrode's (26%). To determine developmental ability in vivo, IVF-derived embryos (n = 586) were transferred at 96 or 120 h to recipients (n = 49) that had undergone synchronous oocyte recovery as donors. The percentage of recipients producing kittens after transfer of embryos cultured for 96 or 120 h in F-10 was 31 and 25, respectively, compared with 55% of 120 h recipients receiving embryos cultured in M-199 or Tyrode's. Overall, more pregnancies occurred following transfer of > or = 12 embryos (11/26) than if < 12 embryos were transferred (6/23).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

[Hormonal therapy for the synchronization between donor and recipient in an oocyte donation and surrogacy fertilization in vitro-embryo transfer program].

OBJECTIVE: To evaluate the efficacy of hormonal therapy for the establishment of endometrium synchronous with the embryo in an oocyte donation and surrogacy fertilization in vitro-embryo transfer (IVF-ET) program. METHODS: The IVF-ET routine of the Beijing Medical University was employed. The recipients included cases with (1) premature menopause and (2) abnormal chromosomal karyotypes; surrogate mothers. For (1) surrogate mother, low constant doses of estradiol (E2) (17-beta estradiol-Estrace or E2 valerate) 2-4 mg was started 3-5 days before the expected menstruation of the donor progesterone (P) was started intramuscularly 30 mg daily from the day of hCG injection, 50 mg from the day of oocyte pick-up, 100 mg from the day of embryo transfer and 150 mg from the day of positive urinary beta-hCG; E2 was maintained at the 4 mg dose. Both E2 and P were continued until after the luteo-placental shift-about 90 to 100 days of pregnancy with gradual tapering doses. The serum E2 and P levels were measured by radioimmunoassay and compared with those of normal pregnancy. For (2) women with normal menstrual periods, the Chinese made gonadotropin-releasing hormone-A (GnRH-A) 400-600 micrograms was given for down-regulation of pituitary and artificial agonadism when at least amenorrhea of two months was achieved and a quiescent state of the endometrium and ovaries was ascertained by vaginal ultrasonography. Then the E2 and P therapy was started as described above. GnRH-A was stopped on the day of hCG injection. RESULTS: In the hormonal therapy group, the serum E2 levels were quite similar to those of the normal pregnancy, while the P levels were about 3-5 times higher. Two full term deliveries of normal female babies and one in her 36th week of pregnancy were achieved in the premature menopause group. In the normal menstruation group, one was in her 15th week of pregnancy and the surrogate mother was in h34 4 1/2 months of pregnancy. CONCLUSION: The proposed hormonal therapy to synchronize the donor and the recipient may be used with satisfactory clinical outcome in an oocyte donation and surrogacy IVF-ET program.

Chorionic Gonadotropin, beta Subunit, Human↗

Alternative embryo transfer on day 3 or day 5 for reducing the risk of multiple gestations.

PURPOSE: This study was carried out to reduce the possibility of high-order multiple gestations and the failure of embryo transfer by determining their replacement date based on the number and quality of 2-day embryos. METHODS: All zygotes were cocultured with cumulus cells in 10 microliters of YS medium containing 10% human follicular fluid (hFF) for 48 or 96 hr. In period I, all embryos were transferred on day 3 (1032 cycles). In period II, the embryos were transferred on either day 3 or day 5 by determining their replacement date based on the number and quality of 2-day embryos: there were 2701 patients in whom embryos were replaced on day 3 (in the case that the number of zygotes was less than eight and the number of good-quality embryos was less than three) and 1952 patients less than 40 years old in whom embryos were replaced on day 5 (in the case that the number of zygotes was eight or more and/or the number of good-quality embryos was three or more). On the other hand, patients who were 40 years old or more were alloted to day 3 transfer cycles, regardless of the number and quality of the 2-day embryos, due to the possibility of their not producing blastocyst-stage embryos in vitro. RESULTS: The number of embryos transferred in period II was 2.9 +/- 0.6, while that in period I was 3.7 +/- 0.5. The multiple pregnancy rate was significantly decreased in period II (30.7%) compared to that (49.6%) in period I, while the pregnancy and implantation rates in period II (36.1 and 16.4%, respectively) were not lower than those (34.9 and 16.1%, respectively) in period I. The rate of triplet or more gestations was significantly minimized in period II (2.3%) compared to that in period I (26.5%). CONCLUSIONS: We propose that determination of the date on which embryos should be transferred based on the number and quality of embryos on day 2 may help to maintain an acceptable pregnancy rate, while minimizing embryo transfer failure and high-order multiple gestations.

Adult↗

Firm embryo transfer catheters for assisted reproduction: a systematic review and meta-analysis using direct and adjusted indirect comparisons.

A systematic review of published evidence on firm embryo transfer catheters is presented. Extensive searches were conducted for full-text manuscripts, abstracts, ongoing and unpublished trials. Direct and adjusted indirect comparisons were undertaken, where appropriate. Twenty-six randomized controlled trials comparing embryo transfer catheters were identified. Only two trials (314 transfers) compared different firm embryo catheters. Using direct comparison, both the Tom Cat and Tefcat catheters demonstrated statistically significant increased chances of clinical pregnancy compared with the Tight Difficult Transfer (TDT) catheter (P=0.007; OR=3.67, 95% CI=1.48-9.10 and P<0.0001; OR=4.71, 95% CI=2.34-9.48 respectively). The implantation rates were also higher with the Tom Cat and Tefcat catheters than the TDT catheter (P=0.005; OR=3.67, 95% CI=1.48-9.10 and P<0.00001; OR=4.29, 95% CI=2.45-7.50 respectively). Using adjusted indirect comparison, Tom Cat and Tefcat catheters were compared, and shown to have similar pregnancy and implantation rates (OR=0.99; 95% CI=-0.87-1.79 and OR=0.86; 95% CI=-0.77-1.35). In conclusion, both Tom Cat and Tefcat catheters give better outcomes than the TDT catheter, but are similar to each other.

Catheterization↗

Low-dose glucocorticoids after in vitro fertilization and embryo transfer have no significant effect on pregnancy rate.

OBJECTIVE: To determine the effect on pregnancy rate (PR) of low-dose glucocorticoid treatment in cycles without micromanipulation. DESIGN: Randomized, prospective, double-blinded, placebo-controlled trial. SETTING: One university-based tertiary infertility center and two private infertility centers. PATIENTS: All patients receiving standard stimulation IVF-ET or transfer of cryopreserved embryos at the participating facilities from January to September 1993 were asked to participate in this study. Patients having micromanipulation were excluded from this study. INTERVENTIONS: Participating patients were randomized to either 16 mg oral 6-alpha-methylprednisolone for four evenings starting the evening of retrieval or the evening before thawing cryopreserved embryos or to placebo administered in an identical fashion. Both groups were treated with 250 mg oral tetracycline four times per day starting with initiation of the study medication and continuing for 4 days. Cryopreservation and stimulation cycles were managed according to pre-established protocols for all patients. A clinical pregnancy was confirmed by an appropriately rising hCG titer and a gestational sac on ultrasound. RESULTS: A total of 206 stimulation patients and 61 cryopreservation patients were randomized and had an ET. Patient characteristics were similar between groups. The clinical pregnancy and implantation rates between placebo and glucocorticoid groups were 35.9% versus 40.8% and 12.8% versus 11.7% for stimulation cycles and 30.3% versus 25% and 9.9% versus 7.4% for cryopreservation cycles, respectively. None of these differences were statistically significant. CONCLUSIONS: Glucocorticoid plus antibiotic treatment at these doses for transfers of nonmicromanipulated embryos does not appear to have a significant effect on pregnancy or implantation rates.

Adult↗

In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1990 results from the IVF-ET Registry. Medical Research International. Society for Assisted Reproductive Technology (SART), The American Fertility Society.

OBJECTIVE: To summarize the procedures and outcomes of assisted reproductive technology (ART) initiated in the United States in 1990. DESIGN: The registry follows a prospective study format. PARTICIPANTS: One hundred eighty clinics submitted data on their ART treatments to the In Vitro Fertilization (IVF) Registry. MAIN OUTCOME MEASURES: The outcomes measured included clinical pregnancy, ectopic pregnancy, abortion, stillbirth, delivery, chromosomal abnormalities, and congenital defects. RESULTS: During 1990, the clinics reported performing 25,744 ovarian stimulation cycles. From all ART treatments there were 5,150 clinical pregnancies and 3,951 live deliveries. The overall live delivery rates were 14% for IVF (based on 16,405 retrievals), 22% for gamete intrafallopian transfer (based on 3,750 retrievals), and 16% for zygote intrafallopian transfer and related practices (based on 1,370 retrievals). The delivery rates for frozen embryo transfer cycles and IVF with donor egg cycles were 9% and 22%, respectively. CONCLUSIONS: The 1990 results indicate an increase in the use of gonadotropin-releasing hormone analogs and an increase in the number of embryos being transferred per cycle, along with no important changes in clinical pregnancy and delivery rates. In addition, there was an increase in both numbers of frozen embryo transfers and IVF cycles with donor oocytes.

Abortion, Spontaneous↗

Prevention of transmission of sheep pulmonary adenomatosis by embryo transfer.

Two-hundred-and-fifteen embryos recovered from 76 donor ewes from flocks endemically infected with sheep pulmonary adenomatosis (SPA) and mated with uninfected rams were transferred to 131 uninfected recipients under strict sanitary conditions using International Embryo Transfer Society protocols. The recipients and their progeny were kept in a closed, isolated SPA-free flock. Thirty-eight of 51 progeny from SPA-positive donors and 55 of 74 progeny from donors in which no lesions of SPA were detected survived for at least five years after birth. In a similar study 11 embryos from four uninfected donors mated to an SPA-infected ram were transferred to seven recipients, and four of five progeny born to four recipients survived for at least five years. No evidence of SPA was found in the recipients or their progeny by embryo transfer in either study. On the basis of clinical and pathological criteria, it is concluded that embryo transfer can be used to provide an effective barrier against the transmission of SPA from donors from infected flocks, whether or not the parents show clinical signs of the disease.

Animal Husbandry↗

Occurrence of cleft palate, palatal slit, and fetal death in mice treated with a glucocorticoid: an embryo transfer experiment.

SWV and C57BL/6 (C57BL) mice were treated subcutaneously with triamcinolone acetonide in a single dose of 2.5 mg/kg on day 12 of pregnancy (vaginal plug = day 0), and the palate of their fetuses was examined at term. Cleft palate was seen in some SWV and C57BL fetuses; its frequency was significantly higher in the former. Closer examination revealed palatal slit in some C57BL, but in no SWV fetuses. In addition, fetal mortality was significantly increased in SWV, but not in C57BL, exposed to triamcinolone. These strain differences in cleft palate, palatal slit, and fetal mortality were investigated by embryo transfer. The results showed that, in cleft palate induction, the effects of uterine environment were more important than those of fetal genotype. On the other hand, after transfer, palatal slit still occurred in C57BL but not in SWV fetuses; thus, in palatal slit occurrence, the fetal genotype played a more important role than the uterine environment. Accordingly, it is suggested that the nature of the participation of fetal genotype and uterine environment in palatal slit occurrence is different from that in cleft palate induction. In regard to fetal mortality, embryo transfer procedures influenced it in SWV dams and the effect of triamcinolone could not be detected after embryo transfer.

Abnormalities, Drug-Induced↗

Effect of bladder filling on embryo transfer.

We have investigated a claim that bladder filling, by straightening an anteverted uterus into line with the vagina, might facilitate embryo transfer through the cervix after in vitro fertilization. In a randomized controlled trail in 66 women with and 76 without a filled bladder, we found no significant differences in difficulties encountered at embryo transfer or in the chance of pregnancy.

Adult↗

The characterization of bovine embryos obtained from prepubertal calf oocytes and their viability after non surgical embryo transfer.

We have previously shown that the addition of epidermal growth factor (EGF) during in vitro maturation was capable of stimulating the cytoplasmic maturation of cow and calf oocytes. The aim of the present study was to compare calf and cow blastocysts produced in the presence of EGF in terms of total cell number and cell distribution between trophectoderm (TE) and inner cell mass (ICM), pattern of protein synthesis, and ability to establish pregnancy after embryo transfer to recipients. For all experiment, embryos at Day 7 were obtained from IVM/IVF/IVC oocytes. No significant differences were noted in total cell number (cow = 138 +/- 46 vs calf = 142 +/- 59; mean +/- SD) or ICM and TE cell number between calf (ICM = 35 +/- 19, TE = 107 +/- 52) and cow (ICM = 38 +/- 21, TE = 99 +/- 32) blastocysts, nor in the ICM/total cell number ratio (cow = 0.27 +/- 11, calf = 0.25 +/- 12). No differences were noted in the constitutive and the neosynthetic protein profiles between cow and calf embryos obtained in vitro. The results of embryo transfer, showed that there was higher pregnancy loss following transfer of calf compared with cow embryos. After Day 35, the rate of pregnancy decreases, with only 22% of calf embryos maintaining pregnancy until calving compared with 39% for cow embryos. In conclusion, it would seem that embryos originating from calf oocytes are less capable of establishing pregnancies than embryos obtained from adult oocytes, although this difference was not significant. This low viability cannot be explained by differences in cell number or by the protein profiles identified between these 2 groups of embryos.

Abortion, Spontaneous↗

[The analysis of selected performance parameters following embryo transfer in the example of cattle].

Gestation lengths and birth weight of calves from German Red Pied cows were used to analyse differences between embryo transplant offsprings (n = 76) and offsprings from natural pregnancy (n = 118). Furthermore differences were examined in milk traits between embryo transplant (n = 76) and not embryo transplant offsprings (n = 66) from the same dam (German Black Pied, n = 38). After embryo transfer gestation lengths was shorter, but only with a clear difference in first calving cows. Calves from embryo transfer had lower birth weights, because most of the time recipients are maiden. Even the age effect was taken into account, birth weights were about 1.5 kg lower for embryo transplant offsprings. For milk traits no significant differences could be ascertain between embryo transplant and not embryo transplant offsprings. With the exception of fat yield the estimated breeding values of the offsprings correspond to the expectations.

Animals↗

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: effect on developmental potential.

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm.

Animals↗

[The results of a program of in vitro fertilization and tubal embryo transfer (ZIFT)].

Twenty-one sterile couples, between September 1989, and August 1991, were treated for ovulation stimulation of their cycles, in order to practice in them, a in vitro fertilization and tube embryo transfer (ZIFT). Two protocols of ovulation induction were used, both with leuprolide acetate (Lupron), one in the luteal phase and the other in follicular phase and since the second or the fourth day of the cycle, respectively, gonadotropins were added (Metrodine and Pergonal). Out of all the twenty-nine initiated cycles, twenty-seven were aspirated (93.1%) and twenty-four reached an embryo transfer (82.8%). Seven clinic pregnancies were obtained (29.17% per transfer) and four deliveries (16.67% per transfer). The sterility period average was 69.64 +/- 36.6 months and the patients age average was 34.1 +/- 4.38 years. The global rate of fertilization was 63.53%. With luteal phase Lupron best results were got (pregnancy rate of 38.46% per transfer) and there were not considerable difference in the number of gonadotropins ampulla employed. When embryos were transfer to the tubes and the uteri the pregnant rate was 50% per transfer, in comparison to 18.75% when transfer was made only in the tubes.

Chile↗