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PubMed1996

Embryo viability, duration of gestation and birth weight in sheep after transfer of in vitro matured and in vitro fertilized zygotes cultured in vitro or in vivo.

The influence of various in vitro procedures on embryo survival and the production of normal offspring was investigated in sheep. Zygotes produced from in vitro matured (IVM) and fertilized (IVF) oocytes derived from slaughtered Merino ewes were allocated to three culture treatments for 6.5 days. Two groups were cultured in vitro in the absence or presence of oviduct epithelial cells while the third group was cultured in vivo in the oviducts of synchronized ewes. A fourth group of zygotes obtained from superovulated Merino ewes was also cultured in vivo and served as controls. After culture, IVM-IVF morulae and blastocysts, and control embryos were transferred to final recipient ewes. Pregnancy was diagnosed at day 50 of gestation by ultrasonography and pregnancies were allowed to go to term. The survival to term of IVM-IVF zygotes cultured in vitro was reduced compared with both in vivo cultured IVM-IVF zygotes and control zygotes (25-35% versus 51-60%, respectively, P < 0.05). Day 6.5 IVM-IVF morulae had a lower survival rate than did control morulae regardless of culture treatment (P < 0.05), while survival rates of day 6.5 IVM-IVF blastocysts cultured in vivo did not differ from those of control blastocysts (P > 0.1). Both the gestation period and birth weight of IVM-IVF lambs were increased when compared with controls, the former significantly in all groups (154.0-154.9 days versus 150.6 days; P < 0.01), while the latter increase was on the borderline of significance (4.5-4.8 kg versus 4.0 kg; 0.01 < or = P < or = 0.1, respectively) and dependent on the prolongation of the gestation period. It is concluded that in vitro maturation and fertilization compromise subsequent embryonic and fetal development in sheep irrespective of the subsequent in vivo or in vitro culture treatment. Subjecting IVM-IVF zygotes to in vivo culture for 6.5 days minimizes only some of these effects, thus leading to the aberrant production of some offspring.

Animals↗
PubMed1990

Effects of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro.

The present study was conducted to clarify the effect of heparin dosage and sperm capacitation time on in vitro fertilization (Experiment 1) and cleavage (Experiment 2) rates of bovine oocytes matured in vitro. For in vitro fertilization, seven dosages of heparin (0, 5, 10, 25, 50, 100 and 200 microg/ml) and nine incubation periods (0, 5, 15, 30, 45, 60, 120, 180 and 240 min) in a capacitation medium were examined, using 6,634 oocytes. The mean proportions of fertilized oocytes in 25, 50 and 100 microg/ml of heparin were significantly (P<0.05) higher (53 to 59%) than in the other dosages (3 to 44%). Incubation with heparin for longer than 60 min lowered the frequencies of fertilization (20 to 36%) compared with the shorter incubation periods (38 to 49%). Higher proportions of fertilized oocytes were obtained by 5, 15, 30 or 45 min of incubation (42 to 49%) than by the other time periods (20 to 38%). Cleavage rates were found by using 2,098 oocytes in a factorial study (4x4x15: dosages -25, 50, 100 and 200 mug/ml; incubation periods -0, 15, 30 and 60 min; and replicates). The incubation periods and replicates resulted in highly significant differences (P<0.001) in development rates to eight-cell stage, but the four dosages of heparin showed no significant differences. The present results indicate that heparin dosage and sperm capacitation time are important factors influencing in vitro fertilization and cleavage rates. Optimal heparin dosages for the capacitation of bull spermatozoa ranged from 25 to 100 microg/ml; optimal incubation periods ranged from 5 to 60 min.

Journal Article↗
PubMed1997

Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry.

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

Journal Article↗
PubMed1986

Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes.

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2, and allocated to 3 groups. For Groups I and II, unmated donors were killed 67-69 h after PMSG injection, shortly after the expected time of ovulation. Oocytes were recovered from the oviducts and transferred immediately into the oviduct of mated recipients (Group I) whose ipsilateral ovary had been exposed by peeling back the bursa, preventing endogenous oocytes from entering the oviduct, or were fertilized in vitro (Group II) and were transferred 16-18 h later. Rats in Group III were allowed to mate and half were killed 6 h after mating. The fertilized oocytes were then incubated for 10-12 h until transfer. The remaining rats in Group III were killed 16-18 h after mating and fertilized oocytes were collected and transferred immediately. Recipient rats were killed on Days 2, 5, 8 and 20. Zygotes resulting from in-vitro fertilization (Group II) were as able as those fertilized in donors (Group III) or recipients (Group I) to develop to the 2-cell stage, but underwent significantly greater embryonic loss beyond this stage of development. There was a slower rate of development of such oocytes to the blastocyst stage (Day 5) and a lower mean weight of implantation sites (Day 8). Transfer of zygotes after in-vitro fertilization resulted in a loss of 35% of the embryos at the time of implantation. These results suggest that in-vitro fertilization of rat oocytes leads to defects in the embryos causing a delay in early embryo development and a large number of implantation losses.

Animals↗
PubMed1996

[Ovulation induction in women in the in vitro fertilization program].

In-vitro fertilization presents a method of assisted reproduction where fusion of two gametes (fertilization) occurs in laboratory conditions. In this way part of the process which normally happens in the fallopian tube is being imitated. By ovulation the number of follicles increases, thus increasing the number of oocytes, that is the number of embryos and at the same time increasing chance of pregnancy. Ovulation induction was achieved by: clomiphene citrate (CC), human menopausal gonadotropin (hMG), human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRH). Our trial compared effects of three induction schemes: HMG-hCG; CC-hMG-hCG and LHRH-hMG-hCG. We compared the following parameters: total number of follicles, number of mature follicles (16mm with simultaneous estradiol level 350pg/ml per follicle), number of aspirated oocytes as well as the number of embryos produced by insemination of aspirated oocytes. Our examination revealed that there was no significant difference in number of follicles, aspirated and produced oocytes in patients undergoing stimulated ovulation scheme CC-hMG-hCG in regard to those with hMG-hCG schemes. In patients undergoing LHRH-hMG-hCG scheme the number of follicles was decreased and there were less oocytes, but the number of developed embryos after oocytes insemination was increased. This leads to the conclusion that in-vitro fertilization depends on quality, not only the number of oocytes obtained by LHRH agonists induction.

Adult↗
PubMed1997

Relationship of human follicular diameter with oocyte fertilization and development after in-vitro fertilization or intracytoplasmic sperm injection.

The aim of this work was to evaluate the relationship between follicular size at the time of oocyte retrieval, and the subsequent oocyte competence to be fertilized and to develop in vitro. All the obtained oocytes were classified according to the corresponding volume of aspirated follicular fluid. Aspirated volume of follicular fluid <2 ml corresponded to a follicular diameter <16 mm and constituted the small size group. Volume of follicular fluid from 2 to 6 ml corresponded to a diameter from 16 to 23 mm and constituted the medium size group. The large size group contained follicles with diameter >23 mm and corresponded to an aspirated volume of follicular fluid of >6 ml. A progressive and significant increase in the rates of oocytes with a first polar body was observed from the small size group to the other groups and from the medium to the large size group: 75.3, 85.9 and 95.3% respectively. After classical in-vitro fertilization (IVF), significantly better rates of fertilization and development were obtained in the medium size group compared to the two other groups. Moreover, a positive relationship was observed between follicular diameter and rates of embryos scored as 'good' when oocytes were fertilized by intracytoplasmic sperm injection (ICSI). These results demonstrated that follicular size is positively related to the oocyte ability to be fertilized and to develop. Although oocytes from small follicles gave lower percentages of development probably due to partial oocyte incompetence, they allowed an increase in the total number of embryos scored as 'good'.

Cell Size↗
PubMed1994

The influence of subzonal microinsemination of oocytes failing to fertilize in scheduled routine in-vitro fertilization cycles.

The subzonal microinsemination (SUZI) of oocytes failing to fertilize following routine in-vitro fertilization (IVF) insemination was studied in comparison to routine reinsemination. The data presented indicate that fertilization rate was significantly better with SUZI (23.4%) than with reinsemination (8.3%). The percentage of cleaved replaceable embryos generated was also significantly higher following SUZI than with reinsemination (96.6% versus 66.7% respectively). SUZI led to a significantly higher increase in the percentage of additional number of fertilized oocytes (100%), fertilization cycles (185%), embryos replaced (108%) and embryo replacement cycle over that generated by initial insemination. These were in comparison to 26, 14.3, 11.6 and 15%, respectively, as a result of reinsemination. No pregnancy occurred following replacement of embryos generated from reinsemination. Six pregnancies resulted from replacement of embryos following SUZI. We suggest therefore that SUZI should be the preferred option of practice when oocytes have failed to fertilize following initial insemination for routine IVF.

Embryo Transfer↗
PubMedDate not supplied

The status of the ethics of in vitro fertilization in 1985.

In many respects in vitro fertilization has been accepted as ethical by the majority of people in the Western world. Controversy now involves the ancillary issues of embryo freezing, embryo adoption, surrogate mothering of in vitro fertilization embryos, donor fathering of in vitro fertilization embryos, and research on embryos. The acceptance of in vitro fertilization as ethical was withheld until in vitro fertilization was demonstrated to be safe and effective under controlled circumstances. A similar approach to the ancillary issues is advocated.

Ethics, Medical↗
PubMed1996

A bridge to intracytoplasmic sperm injection--high insemination concentrations benefit patients who have a reduced chance of fertilization with standard in-vitro fertilization.

This study was carried out to determine whether high insemination concentrations (HIC) could improve fertilization and pregnancy rates in patients who had either previously demonstrated poor fertilization rates in vitro using standard protocols (Group 1) or in whom a reduced chance of fertilization was indicated at semen assessment prior to in-vitro fertilization (IVF) (Groups 2 and 3). Forty nine patients were recruited for the study. Standard IVF was carried out in 1 ml volumes using 10(5) spermatozoa/ml. HIC treatment involved co-culture of spermatozoa and oocytes in microdroplets with insemination concentrations increased 10-50 fold higher than standard IVF. Fertilization and pregnancy rates were compared between standard IVF and HIC in individual patients either in consecutive cycles (Group 1) or using sibling oocytes in the same cycle (Group 2). Group 3 patients were treated with HIC for their first treatment cycle. HIC significantly improved the fertilization rate compared with standard IVF for Groups 1 (59.7 +/- 10.7 versus 19.6 +/- 5.4% respectively) and 2 (54.9 +/- 8.5 versus 34.0 +/- 8.5% respectively). HIC increased the pregnancy rate from 0% with standard IVF to 20% per embryo transfer in Group 1 patients. A single pregnancy derived from the transfer of HIC and IVF embryos occurred in Group 2. The fertilization rate (47.2 +/- 7.6%) and pregnancy rate (31.3% per embryo transfer) for Group 3 patients was higher than predicted. There was no increase in the rate of polyploidy with HIC. Provided there are sufficient numbers of motile spermatozoa, HIC may be offered as an initial form of treatment, thus permitting referral of only the poorest responders for intracytoplasmic sperm injection (ICSI).

Adult↗
PubMed1981

Inhibition of in vitro fertilization of mouse eggs: 3-quinuclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa.

The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.

Animals↗
PubMed1992

Successful use of the sperm motility enhancer 2-deoxyadenosine in previously failed human in vitro fertilization.

The outcome of in vitro fertilization treatment in male-factor infertility is generally poor, due mainly to poor fertilization rate and hence fewer available embryos for replacement. This study was carried out to assess the value of asthenospermic sperm exposure to a motility enhancing agent, 2-deoxyadenosine (2-DXA), on our in vitro fertilization program in couples undergoing repeat treatment after previous failed fertilization. Following sperm wash and incubation in 2-DXA-supplemented medium, marked significant improvements were observed in the sperm motility pattern and the number of recoverable sperms as compared to control unexposed samples. There was also a significantly better fertilization rate and higher number of replaceable embryos available following sperm wash in 2-DXA as compared to those washed without it. Following embryo transfer, pregnancy rate was comparable to the generally reported pregnancy rates for routine in vitro fertilization and embryo transfer treatment. Three normal babies were born. Our results indicate that 2-DXA enhances fertilization rate in some asthenospermic patients and that it has no adverse effect on embryonic development.

Adult↗
PubMed1983

[Ovarian stimulation in an in-vitro fertilization programme].

The in-vitro fertilization of human ova and the subsequent transfer of the embryo are a possibility for infertile patients with damaged fallopian tubes to achieve a pregnancy. The results of different methods of ovarian stimulation in an in-vitro fertilization programme are described. The Clomiphen/hCG treatment and the overlapping Clomiphen/hMG/hCG treatment were used. Between July 1982 and March 1983, 130 patients had a laparoscopic aspiration of ovarian follicles at the Department for Obstetrics and Gynaecology in Luebeck. 585 follicles were aspirated and 404 ova retrieved. 210 showed the criteria of a preovulatory ovum. Of these 210, 177 were fertilized. In 95 patients an embryo transfer at the 2-8 cell stage was carried out. Inspite of the good fertilization rate of over 80% and the good embryo transfer rate of over 70%, pregnancy was achieved only in 13 cases. The number of follicles and of the retrieved preovulatory ova as well as the fertilization rate were best in the group which received combined Clomiphen and hMG. The pregnancy rate was not different compared to the other group. The first delivery of a healthy infant following laparoscopic aspiration of a follicle and in-vitro fertilization and embryo transfer in Luebeck occurred in March 1983.

Adult↗
PubMed1982

[In vitro fertilization--a therapeutic procedure?].

In vitro fertilization and subsequent implantation of the embryo is one way of treating infertility in women not only with extensive damage of the Fallopian tubes of inflammatory or post-surgical aetiology, but also with idiopathic sterility. However at our present state of knowledge in vitro fertilization should not be seen as a final answer, separated from other methods of treatment (e.g. microsurgery of the tubes). From the ethical point of view, specific treatment for disorders of the reproductive organs, to which in vitro fertilization belongs, must be permitted.

Embryo Transfer↗
PubMed1996

Rederivation of mice by means of in vitro fertilization and embryo transfer.

In vitro fertilization and embryo transfer were performed for rederivation of four strains of mice harbouring mouse hepatitis virus (MHV) and/or Pasteurella pneumotropica (P. pneumotropica). Superovulated oocytes were fertilized by preincubated cauda epididymis sperm in vitro. Fertilized eggs at 2-cell stage were transferred into the oviducts of specific pathogen free (SPF) recipients. Microbial examination of sperm and/or oocyte donors verified the presence of P. pneumotropica and/or of antibodies to MHV in all strains, but neither in the recipients nor in the offspring antibodies to MHV could they be detected. The results indicate that an in vitro fertilization-embryo transfer (IVF-ET) system is an effective and simple alternative to cesarean operation in infected mice.

Animals↗
PubMed2000

Obstetric outcome and follow-up of children born after in vitro fertilization (IVF).

In vitro fertilization (IVF) is a well established and effective method for the treatment of infertility, but there is concern about the health of children born as a result of this procedure. The introduction of new technologies, such as intracytoplasmic sperm injection (ICSI), has increased concern that the offspring from such techniques may be at increased risk, particularly of malformations. Studies on obstetric and neonatal outcome and early infant development after IVF obtained from a Medline search were reviewed. Children born after IVF had a considerably higher risk of being born pre-term and with a lower birth weight than children conceived naturally. A high incidence of multiple births and maternal characteristics were the main factors responsible for the increase in adverse outcome. Novel strategies in assisted reproduction, including the development of single embryo transfer regimens and avoidance of multiple births, are required. There is also a need for further developmental follow-up of children born after assisted conception, especially those born after ICSI.

Journal Article↗
PubMed1998

Case report: high fertilization rate in conventional in-vitro fertilization utilizing spermatozoa from an oligozoospermic subject presenting microdeletions of the Y chromosome long arm.

A case is reported in which a high fertilization rate was achieved by conventional in-vitro fertilization (IVF), using spermatozoa from an oligozoospermic man carrying a microdeletion of the long arm of the Y chromosome. The patient presented with idiopathic infertility of 10 years duration; the fertility status of his wife was completely normal. After IVF, five out of eight oocytes retrieved showed normal fertilization and four showed normal embryo cleavage. Four embryos were transferred; however, pregnancy did not result. These results demonstrate that spermatozoa from oligozoospermic patients carrying a Yq microdeletion are fully competent in achieving capacitation, acrosome reaction and fertilizing ability during IVF. Therefore, although definitive conclusions cannot be made from a single case report, we suggest that Yq microdeletion analysis should be considered in oligozoospermic patients undergoing conventional IVF.

Adult↗
PubMed2006

Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C.

Sperm recovery from the cauda epididymis can be very advantageous, for example, in case of the unexpected death of a genetically highly valuable animal, for preserving endangered species, or when the collection of sperm by other means becomes impossible. Studies indicate that epididymides stored at cooler temperatures result in better quality sperm. One of the factors that could negatively affect sperm viability during storage is lipid peroxidation, in which the sperm membrane's ability to resist attacks by reactive oxygen species (ROS) plays an important role. Another factor is the presence of cytoplasmic droplets, which appear in high numbers in epididymal sperm, and are known to influence oxidative stress. The objectives of this study were: to determine whether the post-slaughter storage temperature of the epididymis would effect the sperm membrane's resistance to lipid peroxidation and/or the sperm cell's fertilizing capacity in vitro and to elucidate the role played by the cytoplasmic droplets. Forty-eight testicles with epididymides (24 bulls) were collected following slaughter, and divided into two groups. One testicle from each pair was stored at 4 degrees C, and the other at 34 degrees C, for 2h, after which sperm was collected from the caudae epididymides. Sperm concentration was measured, and an aliquot containing 10(8)sperm was subjected to induced lipid peroxidation with ferrous sulphate and ascorbate (37 degrees C, 2h). Subsequently, thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, were measured. A second aliquot of the same sample was used in a routine in vitro fertilization performed in duplicate. Sperm from caudae epididymides stored at 34 degrees C resulted in lower rates of total blastocyst formation and had a higher percentage of distal droplets, when compared to sperm from epididymides stored at 4 degrees C (21.2+/-2.42 and 71.8+/-4.7% versus 33.5+/-1.8 and 23.7+/-4.7%, respectively, P<0.05). Storage temperature had no effect on TBARS levels. For samples stored at 4 degrees C, TBARS were negatively correlated with distal droplets (r=-0.63, P<0.05) and positively correlated with proximal droplets (r=0.42, P<0.05). In conclusion, our results show that short-term storage of epididymides at 4 degrees C provided sperm of higher quality and in vitro fertilizing capacity than storage at 34 degrees C. Although resistance to oxidative stress could not be shown to directly influence these results, distal sperm droplets that appeared in high numbers in the cooled epididymal sperm samples, may have exerted an antioxidant effect. We hypothesize that this protection against ROS is one of the functions of distal sperm droplets in the epididymis.

Animals↗
PubMed1984

Morphology and X-ray microprobe analysis of spermatozoa from fertile and infertile men in in vitro fertilization.

This paper reports differences observed in the elemental content of fertile and infertile human spermatozoa used in an in vitro fertilization (IVF) program. "Fertile" and "infertile" were designated by the successful penetration or failure to penetrate an oocyte in vitro. We report morphological and morphometric differences which, together with elemental changes, may be causes of infertility in apparently normal spermatozoa. There were significant differences (P less than 0.05) in sodium and chlorine concentrations between fertile and infertile samples and there was more chlorine than could be accounted for as sodium chloride. Many spermatozoa showed particles adhering to tails, with a higher incidence of "contamination" in the infertile spermatozoa. There were significant differences in both shapes of heads and lengths of tails between fertile and infertile spermatozoa.

Chlorine↗