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At least 1,189 records · Page 66Linked to original sources
PubMed1996

Classification of male factor infertility relevant to in-vitro fertilization insemination strategies using mannose ligands, acrosome status and anti-cytoskeletal antibodies.

Polyvalent mannose ligands in the presence of free mannose act as zona pellucida agonists which rapidly induce acrosome exocytosis in competent motile human sperm from fertile donors following in-vitro capacitation. Quantification of the binding patterns of fluorescein isothiocyanate-labelled mannosylated albumins and of specific antisera which recognize mannose receptors and other related integral sperm membrane proteins as well as the incidence of induced acrosome exocytosis after capacitation has allowed us to identify three categories of male infertility. Category 1 males have normozoospermic semen parameters, their spermatozoa have elevated sperm cholesterol values and fail to fertilize oocytes in vitro after standard short-term incubations. These spermatozoa do not bind mannose ligands and do not show spontaneous or induced acrosome reactions, but treatments to remove cholesterol from the spermatozoa (e.g. prolonged incubation in the presence of sterol acceptors) confer the ability to fertilize. Cholesterol loading and unloading experiments have demonstrated the reversible character of sperm membrane properties in category 1 male infertility. Category 2 males have normal-appearing spermatozoa in semen which express mannose ligand receptors on incubation, but fail to undergo acrosome reactions in response to mannose treatment. Interestingly, all category 2 males identified in this study have clinical varicocele. Category 3 males have semen which may be normozoospermic or teratozoospermic with, in some cases, high percentages of tapering spermatozoa in the absence of clinical varicocele. Spermatozoa from category 3 men are deficient in a superfamily of integral membrane proteins whose cytoplasmic tails have myosin motors as identified by amino acid sequence analysis and anti-myosin antibody reactivity. Their spermatozoa do not express mannose ligand receptors or undergo induced acrosome reactions. Fertilization with category 2 and 3 semen is only achieved by micromanipulation procedures. These findings illustrate the practical application of basic research for infertility classification.

Acrosome↗
PubMed1995

Pregnancy and birth after intracytoplasmic sperm injection with spermatozoa from a patient with tail stump syndrome.

Tail stump syndrome, which may be associated with primary ciliary dyskinesia, is also associated with morphological defects of the flagellum resulting in severe asthenozoospermia. Until recently, these morphological anomalies caused definite male infertility. Today, however, new methods such as micromanipulation techniques provide a rational therapy for this patient group. A pregnancy followed by living offspring was achieved following the intracytoplasmic injection of immotile spermatozoa from a patient with tail stump syndrome.

Adult↗
PubMed1989

Human fertilization by micro-injection of immotile spermatozoa.

Microfertilization of human oocytes with spermatozoa from a man with immotile cilia syndrome is reported, confirming a preliminary investigation where a zona-free donor oocyte was fertilized with spermatozoa from the same patient. Oocytes from his spouse were obtained by laparoscopy after routine stimulation with clomiphene citrate, human menopausal and chorionic gonadotrophins, and were cultured for 4-6 h in Whittingham's T6 medium, supplemented with 10% of her serum. The spermatozoa were washed and processed in the same medium and capacitated for 6-8 h before micromanipulation. Three of five mature oocytes were fertilized by micro-injection of a single immotile spermatozoon into the perivitelline space. One oocyte produced a two-pronuclear ovum assessed 19 h after injection, while the other two produced 2-cell embryos with blastomeres of equal size, 22 h after injection. These embryos cleaved to 3-8-cell stages in culture before embryo replacement. No pregnancy resulted from embryo transfer. The results conclusively demonstrate that human oocytes can be fertilized successfully with immotile spermatozoa by micro-injection and the work has profound implications in the treatment of severe male infertility.

Adult↗
PubMed1989

Developmental control of the human male pronucleus by ooplasmic factors.

The nature of oocyte cytoplasmic factors controlling the development of the male pronucleus was investigated by inseminating human, zona-free oocytes at metaphase of the 1st and 2nd meiotic division. Oocytes at metaphase of the 2nd meiotic division could support the full structural and functional development of male pronuclei, whereas the vast majority of those at metaphase of the 1st meiotic division failed to do so. This suggests that oocyte cytoplasmic factors required for male pronuclear formation do not develop fully until the oocyte reaches the 2nd meiotic metaphase. When increasing numbers of spermatozoa entered one oocyte, the transformation of sperm nuclei into pronuclei was impaired progressively. The later stages of pronuclear development were particularly sensitive to polyspermy. These factors should be taken into consideration in the development of techniques of micromanipulation-assisted insemination.

Autoradiography↗
PubMed1990

Trophectoderm biopsy in human blastocysts.

Trophectoderm biopsy was carried out on 47 human blastocysts. A slit was made in the zona pellucida opposite the inner cell mass by micromanipulative techniques. The human blastocyst zona offered more resistance to slitting compared to that of the mouse. After 18-24 h, controlled herniation of the trophectoderm cells was observed. These cells were biopsied when the diameter of the herniation was approximately equal to that of the blastocyst. The size of the slit and the stage of embryonic development at which slitting was performed were important for successful herniation to occur. After slitting, 76% of day 5-6 blastocysts showed herniation whilst only 42% of day 7-8 blastocysts herniated. Further development of the manipulated embryos was not apparently impaired, as hatching occurred in 44% of the former and 20% of the latter, compared with 18.1% in non-manipulated controls. The biopsied cells (approximately 10-30) usually remained in a clump but 14% formed vesicles on the day after biopsy. There was, however, no evidence of adherence to the dish or formation of monolayers. These results demonstrate the feasibility of trophectoderm biopsy in human blastocysts and that sufficient extra-embryonic material can be obtained by this technique for preimplantation diagnosis of genetic disorders.

Biopsy↗
PubMed1990

Analysis of the first polar body: preconception genetic diagnosis.

In women who are heterozygous for a genetic disease, genetic analysis of the first polar body allows the identification of oocytes that contain the maternal unaffected gene. These oocytes can be fertilized and transferred to the mother without risk of establishing a pregnancy with a genetically abnormal embryo. We have demonstrated that removal of the first polar body has no effect on subsequent fertilization rates or embryonic growth to the blastocyst stage. We have developed a PCR technique to successfully analyze the PI type Z and PI type M genotypes of alpha-1-antitrypsin deficiency and applied this technique for a couple at risk for PI type ZZ alpha-1-antitrypsin deficiency. After standard IVF treatment to stimulate multiple follicle development, eight oocytes were aspirated transvaginally. Polar bodies were removed by micromanipulation from seven oocytes and fertilization occurred in six cases. PCR analysis was successful in five oocytes. One was PI type M, two were PI type Z and two were heterozygous MZ due to crossing over. Embryos from the two oocytes containing the unaffected gene (polar body PI type Z) were transferred in the same cycle 48 h after insemination. No pregnancy was established. The accuracy of the polar body diagnosis was confirmed by polymerase chain reaction (PCR) analysis of an oocyte that failed to fertilize.

Base Sequence↗
PubMed1991

Microsurgical fertilization and teratozoospermia.

Semen parameters were correlated with the outcome of partial zona dissection (PZD) in 42 couples with male factor infertility. Although fertilization rates were reduced, 12% of the embryos implanted following replacement. Spermatozoa from teratozoospermic sperm populations were able to fuse with oocytes following zona penetration through the artificial gaps. PZD followed by insemination with less than 5% normal spermatozoa led to 20 embryos which, upon replacement, did not implant. Motility and sperm count were not clearly correlated with the outcome of PZD and are therefore less useful indicators for patient selection. Teratozoospermic patients who previously failed to fertilize were compared to a group of similar patients who had not attempted IVF before. Although fertilization was significantly improved in first-time patients, 41% of the patients whose spermatozoa were initially unable to fertilize had at least one embryo when PZD was performed. Several pregnancies were established in this group. Subzonal sperm insertion (SZI) and PZD were compared in 19 patients using sibling oocytes. A significant fraction of spermatozoa from infertile men were able to fuse with the oolemma when directly inserted into the perivitelline area. Using a sucrose solution to shrink the ooplasm, only 1% of the oocytes were damaged during SZI. Monospermic fertilization rates following PZD and SZI were 15 and 16%, respectively. Both micromanipulation methods were successful in most patients. However, in two small groups of patients, only one technique resulted in fertilization.

Female↗
PubMed1991

Normal survival and in-vitro development after cryopreservation of zona-drilled embryos in mice.

The importance of the zona pellucida on survival after freezing and thawing was investigated. Zona-drilled and zona-intact mouse embryos were fertilized in vitro, cultured to the 2, 4 and 8-cell stages and frozen using conventional methods. Zona drilling did not affect the survival or development of frozen embryos to the blastocyst stage in vitro. We conclude that partial damage to the zona pellucida during micromanipulation procedures is compatible with rates of survival and development which are not different to those observed in zona-intact control embryos.

Animals↗
PubMed1991

The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro.

Micromanipulation of human oocytes and embryos has provided new opportunities for both the treatment of infertility and the preimplantation diagnosis of genetic disease. It is important to determine whether manipulated embryos develop normally in vitro, as an indication of their suitability for transfer. However, at present there is little information on the development of non-manipulated embryos in vitro for comparison. We have therefore monitored morphological changes and human chorionic gonadotrophin (HCG) secretion in 36 non-manipulated human embryos, including 26 blastocysts and 10 cavitating morulae, daily from day 3 to day 14 of culture. Hatching was observed in 10 (38.5%) blastocysts and five of these adhered to the culture dish and appeared viable until day 14. The secretion of HCG was first detected on day 8, peaked at day 10 (51.11 +/- 8.7 mIU/ml) and then declined but was still detectable in four blastocysts on day 14. There was no overall difference in HCG secretion by hatched blastocysts and those which remained within the zona. However, those hatched blastocysts which showed adherence had significantly increased (P less than 0.05) HCG secretion. For individual blastocysts, the pattern of HCG secretion correlated well with the assessment of morphology. These data provide the basis for comparative studies of morphological changes and HCG secretion in manipulated embryos.

Blastocyst↗
PubMed1991

Decompaction and biopsy of late mouse morulae: assessment of in-vitro and in-vivo developmental potential.

The present study reports a biopsy technique on decompacted late mouse morulae. Zygotes were collected from hyperstimulated F1 hybrids (C57BL6j females x CBAca males) and cultured in modified Earle's balanced salt solution supplemented with 0.5% bovine serum albumin until the late morula stage (92 h post administration of human chorionic gonadotrophin). Decompaction was obtained by exposure of the embryos to Ca(2+)-Mg(2+)-free phosphate-buffered saline (PBS) or an aqueous solution of ethylene-diaminetetraacetic acid (EDTA) and glycine. Using micromanipulation, a single blastomere was aspirated without removal or softening of the zona. The impact of the different decompaction procedures and the biopsy technique was studied by vital staining, by in-vitro culture up to the early egg-cylinder staged and by the recovery of living mice after transfer to pseudopregnant foster mothers. Our investigations revealed no impact of decompaction and biopsy on immediate viability, as assessed by fluorescein diacetate staining. A significant reduction (P less than 0.05) in the number of mouse morulae that reached the early egg-cylinder stage in vitro was observed after the biopsy procedure. We observed that after this microbiopsy technique, successful pregnancies can be obtained but at a lower percentage compared to controls (P less than 0.01).

Animals↗
PubMed1992

Successful fertilization and pregnancy outcome in in-vitro fertilization using cryopreserved/thawed spermatozoa from patients with malignant diseases.

Cryopreservation of spermatozoa before treatment is the only proven effective method available to circumvent the sterilizing effect of therapy in some patients with malignant diseases. Because of impaired sperm quality after freezing and thawing in-vitro fertilization/embryo transfer (IVF/ET) was indicated in 10 patients (12 cycles) during 1986-1990. The patient's mean age was 33.4 +/- 1.6 years. The following diagnoses were made: seminoma (1), testicular carcinoma (3), leiomyosarcoma of the prostate (1), Wegener's granulomatosis (1), non-Hodgkin's (1) and Hodgkin's lymphoma (3). When motile spermatozoa could be recovered after thawing, the total fraction of motile spermatozoa after swim-up separation ranged from 0.2 to 4.2 x 10(6) spermatozoa/ml (eight patients, nine cycles). In all these cases, insemination was performed with multiple oocytes per dish. Fertilization was achieved when swim-up recovered a mean of 1.8 +/- 0.5 x 10(6) spermatozoa/ml and when insemination was performed with at least a calculated concentration of motile spermatozoa of 1 x 10(5) spermatozoa/oocyte. The fertilization rate of preovulatory oocytes was 60%. Four patients achieved a pregnancy: two of them delivered a single healthy baby, one delivered triplet healthy babies and one had a preclinical abortion. In two patients (three cycles), no motile spermatozoa were recovered after thawing, and micromanipulation of oocytes for assisted fertilization was performed. Although fertilized oocytes were transferred, those couples did not achieve a pregnancy. Patients with lymphopathies had the best results, whilst those with testicular neoplasms had the poorest outcome, thus suggesting a poor gametogenic function in the non-affected testis. These results give hope to some patients with malignant diseases to maintain their reproductive capacity through sperm banking and IVF/ET.

Adult↗
PubMed1993

Microsurgical correction of partially degenerate mouse embryos promotes hatching and restores their viability.

We have evaluated the effects of degeneration of blastomeres on the developmental fate of mouse embryos. Micromanipulation techniques were used first to destroy one or two blastomeres of a 4-cell embryo (thereby creating three-quarter and half embryos), and later to repair the anomaly by removing the degenerate material. The embryos were either cultured in protein-free or protein-supplemented medium. When cultured in protein-supplemented medium, three-quarter embryos hatched at the same rate as intact embryos (84 and 91%, respectively), but this rate was reduced (54%; 67/125) when the embryos were cultured in a protein-free environment. Destruction of two blastomeres of a 4-cell embryo and culture in protein-free medium was detrimental, as only 3.2% (4/124) hatched. By supplementing the culture medium with protein, some of these half embryos were rescued, as shown by a 34% (32/95) hatching rate. A more dramatic increase in hatching was achieved, however, after repair of the half embryos by microsurgical removal of the degenerate material. In this case, 72% (78/109) of the repaired embryos were able to hatch. These findings may have implications for human in-vitro fertilization where partial embryonic degeneration or fragmentation often leads to embryonic demise and reduced implantation. Moreover, these observations may provide important clues to mechanisms of mammalian embryonic hatching.

Animals↗
PubMed1993

Zona opening with 308 nm XeCl excimer laser improves fertilization by spermatozoa from long-term vasectomized mice.

Spermatozoa from long-term vasectomized mice have greatly reduced fertilizing ability in vivo and in vitro, which makes this a useful animal model for male factor infertility. The purpose of this study was to evaluate the 308 nm XeCl excimer laser for opening the zona pellucida to enhance the fertilization rate with spermatozoa from vasectomized males. Inseminating zona-intact (control) oocytes with 5 x 10(6) spermatozoa/ml resulted in only 6% fertilization and 33.3% development to the blastocyst stage; zona-opened oocytes showed significant improvement with 31.5% fertilization, 90% cleavage to the 2-cell stage, and 72.2% blastocyst formation. Out of the 130 oocytes in the experimental group, zona ablation was performed successfully on 127 and only three were damaged. These results suggest that laser micromanipulation for assisted fertilization potentially offers a simplified and precise method for mechanical zona cutting.

Animals↗
PubMed1993

Repeated sperm injection under the zona following initial fertilization failure.

When fertilization fails following micromanipulative under-zona insemination, it is possible to repeat the procedure adding more spermatozoa to achieve fertilization, embryonic development and pregnancy. We report on 18 human in-vitro fertilization cycles where this approach was used. In nine cycles only late-fertilized embryos were available for transfer, and these gave rise to two viable pregnancies (22.2% per transfer). In six cycles, where a mixture of late- and timely fertilized embryos were available for transfer, two viable pregnancies arose (33.3% per transfer). In three cycles no fertilization was achieved even after reinsemination by repeated under-zona insemination.

Embryo Transfer↗
PubMed1993

The ability of the hemizona assay to predict human fertilization in different and consecutive in-vitro fertilization cycles.

The objective of this prospective study was to examine the ability of the hemizona assay (HZA) to predict fertilization outcome of mature, pre-ovulatory oocytes under in-vitro fertilization (IVF) conditions. Since a large number of patients were evaluated over a long period, the power of the HZA to prognosticate fertilization results in the same and subsequent (consecutive) IVF cycles of those same patients was assessed. For IVF, only metaphase II oocytes were used. For the HZA, both fresh oocytes donated by patients at the time of IVF and oocytes recovered from surgically removed ovarian tissue (and salt-stored) were used, and bisected by micromanipulation techniques. Matching hemizonae were co-incubated either with spermatozoa from the patient (test) or from a fertile man (control) for 4 h. The number of spermatozoa tightly bound to the zona was counted. Patients (n = 112) were divided into two groups based on HZA results (expressed as HZA index or HZI): HZI > or = 30% (n = 72) and < 30% (n = 40). The patients with HZI < 30% had significantly lower fertilization rates in both the HZA-IVF cycle and in subsequent cycles compared to patients with HZI > or = 30% (P < 0.03). Linear discriminant analysis indicated the HZA to have a sensitivity of 84%, and positive and negative predictive values of 85 and 70% respectively, for prediction of fertilization outcome in a total of 233 cycles. It was concluded that the HZA is a good predictor of fertilization rate in vitro, and can be used in the IVF setting to supply additional clinical information in male-factor patients.

Female↗
PubMed1993

Subzonal sperm insertion with aged human oocytes from failed in-vitro fertilization attempts: fertilization results and some applications.

This study was undertaken to assess the potential of aged human oocytes from failed in-vitro fertilization attempts as a model for the study of fertilization events after subzonal sperm insertion (SUZI). Criteria of aged oocyte suitability for this purpose were (i) the absence of nuclei, (ii) the presence of a polar body, (iii) the absence of cell division or fragmentation, (iv) marked ooplasmic contraction in hyperosmotic medium, and (v) rapid ooplasmic relaxation after returning into normo-osmotic medium following the micromanipulation. Micro-injection techniques were essentially the same as for SUZI with fresh oocytes. Oocytes that fused with the micro-injected spermatozoa developed pronuclei of typical internal structure. However, the number of pronuclei was often higher than that theoretically expected if each sperm nucleus incorporated into the oocyte gave rise to a single pronucleus. Thus, the use of aged oocytes implies the need for a specific method to assess the frequency of fusion in sperm samples examined. The results suggest that the method described here can be applied in a preliminary diagnostic test before a therapeutic SUZI attempt and in studies aimed at the optimization of sperm treatment protocols to increase the fusion capacity of subzonally inserted spermatozoa.

Acrosome↗
PubMed1993

The in-vitro and in-vivo developmental potential of frozen and non-frozen biopsied 8-cell mouse embryos.

We investigated the in-vitro and in-vivo development of cryopreserved and non-cryopreserved biopsied 8-cell mouse embryos, from which one to seven blastomeres were removed by micromanipulation. The results clearly indicate that the in-vitro and in-vivo development of biopsied 8-cell mouse embryos depended on the number of blastomeres removed: the more blastomeres removed, the greater the effect. When five, six, or seven blastomeres were removed, fewer blastocysts were formed. Furthermore, when these blastocysts were transferred to pseudopregnant females, no living young were formed, indicating the abnormality of these blastocysts. When up to three blastomeres were removed, there was no significant effect on the rate of in-vitro blastocyst formation. Living young were found even after the biopsy of four blastomeres, and after biopsy of only one or two blastomeres, the same percentage of living young was obtained as in the non-biopsied control embryos. Biopsied 8-cell mouse embryos were frozen and thawed in straws with 1,2-propanediol (1.5 M) and sucrose (0.1 M) with slow-freezing and rapid-thawing protocols. The survival after cryopreservation, defined as the percentage of embryos with the same number of blastomeres intact as the number of blastomeres before freezing, was excellent and no different from non-biopsied embryos, independent of the number of blastomeres biopsied. Furthermore, cryopreservation had no further impact on the in-vitro and in-vivo development of the biopsied embryos.

Animals↗
PubMed1994

Early follicular rise of serum progesterone concentration in response to a flare-up effect of gonadotrophin-releasing hormone agonist impairs follicular recruitment for in-vitro fertilization.

A retrospective study of 150 cycles of in-vitro fertilization (IVF) was undertaken to determine the impact of elevated serum progesterone in the early follicular phase of IVF cycles utilizing gonadotrophin-releasing hormone agonist (GnRHa) initiated in the follicular phase. A total of 127 patients identified as being at risk for poor response to stimulation were treated with a flare-up protocol of GnRHa combined with high dose follicle stimulating hormone (FSH). Patients were excluded for severe male factor requiring micromanipulation. Patients were stimulated with GnRHa beginning on cycle day 2, and high dose FSH beginning on cycle day 3. Some 85% of the cycles exhibited a rise of serum progesterone to a peak concentration of > 1.0 ng/ml (range, 1.2-4.2 ng/ml) during cycle days 2-6. When compared to cycles with no demonstrable progesterone rise, cycles with a rise were associated with a significantly decreased ovarian response: more ampoules of gonadotrophin were required (mean 26.8 versus 22.6, P < 0.05), lower peak oestradiol concentration was reached (mean 774 pg/ml versus 1030; P < 0.05), and fewer mature oocytes were harvested (mean 4.6 versus 7.5; P < 0.01). Among the different pregnancy outcomes (clinical pregnancy, no pregnancy, ongoing pregnancy, and miscarriage), there were no significant differences detected in the early follicular progesterone concentrations as measured by peak progesterone, progesterone area under the curve (days 2-6), and day of peak progesterone. The follicular phase initiation of GnRHa can result in significant elevations of serum progesterone in the early follicular phase, which may impair follicular recruitment and overall ovarian response.

Adult↗