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J Smitz

Publications and source records attributed to J Smitz.

At least 37 records · Page 2Linked to original sources
PubMed2001

Improving in vitro maturation of oocytes in the human taking lessons from experiences in animal species.

One to three per cent of infertile women develop severe ovarian hyperstimulation syndrome after superovulation for assisted reproduction treatment (ART). This severe complication can be avoided when oocytes are obtained at an immature stage (germinal vesicle stage) out of small or medium-sized follicles. This hypothesis has been tested in several infertile women, but clinical pregnancies are disappointlingly low. This new approach in ART is still at an experimental phase and this treatment has still to be improved before routine clinical application. Experimental work in animals and humans suggest a beneficial effect in providing a short preliminary pretreatment with follicle-stimulating hormone to select for a developing cohort of follicles. The aspiration of oocyte cumulus complexes is carried out with a short needle applying reduced aspiration pressure. A crucial point is to provide the appropriate culture environment for the immature oocytes. An optimal cumulus-enclosed human oocyte culture system needs to be defined. The composition of the culture medium could be suggested by in vitro work carried out in animal models. As developmental competence is established during the latest phases of oocyte growth and is dependent on the storage of RNA, a prolonged in vitro maturation period (before inducing nuclear maturation) could provide the necessary transcriptional and translational changes. The conditions to achieve this improved cytoplasmic maturation by prolonging the in vitro culture remain to be defined. More objective noninvasive parameters for oocyte maturity are also needed to pursue research in this field.

Animals↗
PubMed2001

Protein synthesis during maturation of bovine oocytes, effect of epidermal growth factor.

The objective of this study was to determine whether epidermal growth factor (EGF) affected protein synthesis in oocytes during maturation. Initially, the effect of EGF on oocyte maturation was examined to ensure that there was a beneficial effect of EGF in the protein-free maturation medium used in these studies. Results showed that the presence of EGF during maturation significantly enhanced cleavage rate and development to the blastocyst stage. Development after maturation in the presence of EGF was similar to that seen in medium containing serum, luteinizing hormone, follicle-stimulating hormone and estradiol. Protein synthesis was examined in immature oocytes and after 16 or 24 h maturation. Oocytes from each group were labelled by incubation for 4 h with 35S-methionine, the proteins were then separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Between 400 and 500 proteins could be separated using this method and marked changes in protein synthesis was observed during maturation. Changes in eight different proteins were observed when protein patterns from oocytes matured for 16 h with and without EGF were compared. These results suggest that EGF plays a physiological role in oocyte maturation and identification of the proteins induced by EGF could be important for improving our understanding of oocyte maturation in vitro.

Animals↗
PubMed2001

Comparison of LH concentrations in the early and mid-luteal phase in IVF cycles after treatment with HMG alone or in association with the GnRH antagonist Cetrorelix.

Luteinizing hormone (LH) is mandatory for the maintenance of the corpus luteum. Ovarian stimulation for IVF has been associated with a defective luteal phase. The luteal phases of two groups of patients with normal menstrual cycles and no endocrinological cause of infertility were retrospectively analysed in IVF cycles. Thirty-one infertile patients stimulated with human menopausal gonadotrophins (HMG) for IVF to whom the gonadotrophin-releasing hormone (GnRH) antagonist Cetrorelix 0.25 mg was also administered to prevent the LH surge (group I) were compared with 31 infertile patients stimulated with HMG alone (group II). Despite differences in the stimulation outcome, luteal LH serum concentrations were similar in the two groups. LH values dropped from 2.3 +/- 1 IU/l on the day of human chorionic gonadotrophin (HCG) administration to 1.1 +/- 0.7 IU/l on day HCG +2 in group I (P < 0.0001) and from 5.1 +/- 3 to 1.2 +/- 1.7 IU/l (P < 0.0001) in group II. In the mid-luteal phase, LH concentrations were low in both groups. Our results suggest that suppressed LH concentrations in the early and mid-luteal phase may not be attributed solely to the GnRH-antagonist administration. Pituitary LH secretion may be inhibited by supraphysiological steroid serum concentrations via long-loop feedback and/or by the central action of the exogenously administered HCG via a short-loop mechanism.

Adult↗
PubMed2001

Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture.

To assess their quality, spindles were analysed in mouse oocytes from pre-antral follicle culture. High or low oxygen tension was present during the last 16 or 20 h post human chorionic gonadotrophin (HCG)/epidermal growth factor (EGF) addition. Most oocytes from pre-antral follicle culture possessed typical anastral spindles with flat poles resembling those of ovulated, in-vivo-matured oocytes of sexually mature mice, while denuded oocytes in-vitro matured to metaphase II (MII) formed significantly longer, slender spindles with pointed, narrow poles. Spindles in oocytes from follicle culture were only slightly shorter and less compact at the equator as compared with those of oocytes matured in vivo. Chromosomes were well aligned at the equator in MII oocytes obtained from follicle culture with high oxygen. Maturation rate was significantly reduced by lowering oxygen tension to 5% O2. Prolonged culture and the presence of only 5% O2 dramatically increased the percentage of MII oocytes with unaligned chromosomes. These observations indicate that sufficient oxygen supply and time of retrieval after initiation of resumption of maturation by HCG as well as the microenvironment and cell-cell interactions between oocytes and their somatic compartment are critical in affecting the oocyte's capacity to mature to MII, to form a functional spindle, and to align chromosomes correctly.

Animals↗
PubMed2001

Ovulation induction disrupts luteal phase function.

Abnormalities in the luteal phase have been detected in virtually all the stimulation protocols used in in vitro fertilization, on both the hormonal and endometrial levels. Supraphysiological follicular or luteal sex steroid serum concentrations, altered estradiol: progesterone (E2/P) ratio, and disturbed luteinizing hormone pituitary secretion leading to corpus luteum insufficiency or a direct drug effect have been postulated as the main etiologic factors. Luteinizing hormone supports corpus luteum function, and low LH levels have been described after human menopausal gonadotropin treatment, after gonadotropin-releasing hormone (GnRH)-agonist treatment, or after GnRH-antagonist treatment. These low luteal LH levels may lead to an insufficient corpus luteum function and consequently to a shortened luteal phase or to the low luteal progesterone concentrations frequently described after ovulation induction. A direct effect of the GnRH agonist or GnRH antagonist on human corpus luteum or on human endometrium and thus on endometrial receptivity cannot be excluded, as GnRH receptors have been described in both compartments. Endometrial histology has revealed a wide range of abnormalities during the various stimulation protocols. In GnRH-agonist cycles, mid-luteal biopsies have revealed increased glandulo-stromal dyssynchrony and delay in endometrial development, strong positivity of endometrial glands for progesterone receptors, decreased alphavbeta3-integrin subunit expression, and earlier appearance of surface epithelium pinopodes. These factors suggest a shift forwards of the implantation window. Progesterone supplementation improves endometrial histology, and its necessity has been well established, at least in cycles using GnRH agonists.

Endometrium↗
PubMed2001

Rapid detection of macroprolactin in the form of prolactin-immunoglobulin G complexes by immunoprecipitation with anti-human IgG-agarose.

Immunoprecipitation with anti-human immunoglobulin G-agarose was evaluated for the detection of prolactin-immunoglobulin G (PRL-IgG) complexes in macroprolactinemic samples from hyperprolactinemic patients (prolactin measured using the automated Elecsys PRL assay; Roche Diagnostics, Mannheim, Germany). Using the polyethylene glycol (PEG) precipitation test on serum samples with PRL above 1000 mIU/l, we detected macroprolactin in 38 out of 175 samples with a recovery below 50%. Gel filtration chromatography on a subset of hyperprolactinemic samples confirmed that macroprolactin was the predominant immunoreactive form of PRL in samples which showed a recovery < or = 50%. In 37 out of 38 samples containing macroprolactin, immunoprecipitation with anti-human IgG (anti-hIgG)-agarose revealed a higher PRL-binding (19.1-91.3%) than in the sera containing monomeric PRL (< 10%). In conclusion, we found that PEG precipitation detected macroprolactin in 21.7% of samples with elevated PRL levels as measured by the Elecsys PRL assay, and that in the vast majority of these samples the presence of PRL-IgG complexes could be demonstrated by immunoprecipitation with anti-hIgG-agarose. In view of the high prevalence of PRL-IgG complexes in hyperprolactinemic patients and the simplicity of the test, immunoprecipitation with anti-hIgG-agarose might play a role in the routine laboratory handling of hyperprolactinemic samples, especially with regards to PRL assays where PEG causes interference.

Antigen-Antibody Complex↗
PubMed2001

Alterations in ovarian function of mice with reduced amounts of KIT receptor.

The KIT receptor, present on oocyte and theca cells in ovarian follicles, and its ligand, KIT LIGAND, produced by granulosa cells, are encoded at the Kit gene and the Mgf gene, respectively. Both Kit and Mgf mutations affect oogenesis and folliculogenesis. In this study, the ovarian function of heterozygous mice with a mutation Kit(W-lacZ) was examined. Firstly, the amounts of KIT and KIT LIGAND proteins in the ovaries of mice at different ages were determined. Secondly, in vivo and in vitro folliculogenesis of wild type and heterozygous mice were compared. Western blotting showed that the amounts of both KIT and KIT LIGAND proteins were decreased in mutant mice. Ovarian follicle populations were counted and more type 5a follicles and fewer type 5b (preantral follicles) were present in ovaries from Kit(W-lacZ/+) ovaries. Furthermore, the relationships between oocyte size and follicle size differed between wild type and heterozygous mice. This finding may be a consequence of altered proliferation of granulosa cells or of altered oocyte growth in mutant mice. Other features of folliculogenesis, such as initiation of follicular growth, total follicle population and follicular atresia, were not affected by the mutation. Analysis of in vitro folliculogenesis did not reveal other differences between wild type and mutant mice. It is concluded that the Kit(W-lacZ) mutation affects the expression of KIT and KIT LIGAND proteins, resulting in alterations in granulosa cell proliferation and/or oocyte growth in preantral follicles.

Animals↗
PubMed2001

Differences in follicular function of 3-month-old calves and mature cows.

After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Müllerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Müllerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation.

Activins↗
PubMed2001

Confocal microscopy: principles and applications to the field of reproductive biology.

Confocal microscopy allows analysis of fluorescent labeled thick specimens without physical sectioning. Optical sections are generated by eliminating out-of-focus fluorescence and displayed as digitalized images. It allows 3-dimensional reconstruction (XYZ) and time-analysis (XYT), thus providing unique chance to link morphology with cell function. Since images are obtained by scanning, excess illumination of the specimen and quick decrease of the fluorescent signal are avoided. Resolution obtained with a Laser Scanning Confocal Microscopy (LSCM) is theoretically better than that of a conventional microscope. The preparation of the specimen may be based on standard techniques, such as immunocytochemistry applied to fixed cells, or on staining of living cells, following the use of different fluorescent probes at the same time (colocalization). In our laboratory, we use the LSCM system Fluoview version 2.1 (Olympus) to study reproductive biology of animals and humans. We work on stainings of oocytes and blastocysts (mouse, bovine, human), and human ovarian tissues. We study mitochondrial distribution, cortical granule migration, calcium oscillations and spindle quality to link culture conditions and oocyte quality. Staining of F-actin is used to check transzonal projections (in zona pellucida) or to detect abnormalities following experimental treatment. Blastocyst quality is analyzed in sequential optical sections for microfilament organization and counting of total cell number (staining with phalloidin (actin) and picogreen (DNA). Trophectoderm and inner cell mass distribution (differential staining), apoptotic cells (TUNEL method) and viable cells (live/dead test) are also evaluated. Confocal imaging can be helpful for rapid determination of follicle density (staining with AM Calcein) and follicle morphology (picogreen) in ovarian cortical biopsies. The current review describes the principles of confocal microscopy and illustrates its applications to the field of reproductive biology by a large collection of pictures.

Animals↗
PubMed2000

Effects of Kit Ligand and anti-Kit antibody on growth of cultured mouse preantral follicles.

Paracrine regulations between the oocyte and granulosa cells are likely to be key regulators of early folliculogenesis. Evidence obtained from genetic mutants as well as in vivo experiments suggest that Kit and Kit Ligand (KL) may regulate early follicular morphogenesis and function. In this study, we used in vitro culture of intact mouse follicles to confirm and extend these findings. Two concentrations of Kit Ligand (20 and 50 ng/ml) or an antibody blocking the Kit-Kit Ligand interactions (SC1494) were added to preantral follicles grown individually for 12 days and which were finally triggered to ovulate. Effects on follicle and oocyte survival, granulosa cell function (antrum formation, cell numbers, steroidogenesis), and oocyte function (growth, survival, nuclear and/or cytoplasmic maturation) were then analyzed. In optimal culture conditions (presence of 5% fetal calf serum), 50 ng/ml of KL significantly improved cytoplasmic maturation of the oocyte and increased follicular testosterone output, but other parameters were not altered. In serum-free culture conditions, KL was mitogenic for granulosa cells at 50 ng/ml, but could not induce antrum formation and no differences were observed between control and treated groups for steroidogenesis or oocyte growth. Blockade of Kit-Kit Ligand interactions by addition of a blocking antibody decreased oocyte survival 6-9 days after addition of the antibody, but did not upset granulosa cell proliferation. Antrum formation was, however, strongly inhibited. In addition, the blocking antibody markedly reduced aromatase activity of granulosa cells. We conclude that Kit/KL interactions are important for antrum formation and follicular steroidogenesis and regulate survival and cytoplasmic maturation of the oocyte.

Animals↗
PubMed2000

Serum inhibin B in normal term-born male and female neonates during the first week of life.

UNLABELLED: Inhibin B, a gonadal peptide regulating follicle stimulating hormone (FSH) secretion in adults, has been found during gestation in amniotic fluid, but at birth only in term cord blood of male babies. Since no data are available on the evolution of serum inhibin B during the 1st week of life, we studied changes in inhibin B using a specific and sensitive immunoassay in male and female neonates during the 1st week of life in relation to FSH and to evaluate the possible effect of perinatal factors on inhibin B production. Inhibin B was measured by a specific monoclonal enzyme-linked immunosorbent assay. Inhibin B was detectable in cord blood of all eight longitudinally studied male newborns, correlated negatively with the ponderal index and increased significantly on day 5 (from 54.2 +/- 18.5 to 100.4 +/- 34.8 ng/l, P < 0.005). Cord blood inhibin B was detected in only 1 out of 13 screened female neonates. In 48 at term-born females in whom inhibin B was measured on the 5th day of life, only 20 cases had a detectable level (between 8 and 68.6 ng/l). Inhibin B concentrations in cord blood and on day 5 were independent of duration of pregnancy, type of delivery, Apgar score and FSH concentration. CONCLUSION: A sexual difference in serum inhibin B is already present at the end of gestation and changes in inhibin B during the 1st week of life are independent of follicle stimulating hormone changes and perinatal factors in both sexes. Our data suggest that neonatal inhibin B could be used to study whether the newborn has functional testes, i.e. in babies with ambiguous genitalia and/or bilateral cryptorchidism.

Age Factors↗
PubMed2000

Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group.

In this prospective and randomized study, 188 patients received the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix, and 85 patients the LHRH agonist buserelin to prevent endogenous luteinizing hormone (LH) surges during ovarian stimulation in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Ultimately, 181 patients (96.3%) in the cetrorelix group, and 77 (90.6%) in the buserelin group, reached the day of the human chorionic gonadotrophin (HCG) injection. The mean number of human menopausal gonadotrophin (HMG) ampoules administered and the mean number of stimulation days with HMG were significantly less in the cetrorelix group than in the buserelin group (P < 0.01). A rise in LH and progesterone concentrations was observed in three of the 188 patients (1.6%) who received cetrorelix. On the day of the HCG administration, more follicles of a small diameter (11-14 mm) were observed in the buserelin group than in the cetrorelix group (P = 0. 02) and the mean serum oestradiol concentration was significantly higher in patients who received buserelin than in those who received cetrorelix (P < 0.01). Similar results were observed in fertilization, cleavage and pregnancy rates in the two groups. In conclusion, the use of the LHRH antagonists might be considered more advantageous because of the short-term application needed to inhibit gonadotrophin secretion, so allowing a reduction in the treatment time in a clinically significant manner.

Adult↗
PubMed2000

Progesterone for the luteal support of assisted reproductive technologies: clinical options.

The role of progesterone in luteal support in assisted reproductive technologies (ART) is reviewed. There is insufficient data in ART treatment without gonadotrophin-releasing hormone (GnRH) analogues, to prove the necessity for luteal phase support using progesterone. Prospective studies have shown that ART cycles using GnRH analogues need to be supplemented. GnRH antagonists could have some adverse effects on the luteal phase. So far, no prospective randomized comparative study has been performed to investigate the necessity for luteal phase support when antagonists are used in ART cycles. Clinical outcome data (pregnancy and abortion rates) show similar success rates for human chorionic gonadotrophin (HCG) or progesterone supplementation. A major disadvantage of using HCG is the risk of maintaining or enhancing ovarian hyperstimulation syndrome. Of the several routes of administration of progesterone, the vaginal route is preferred because of its ease of use, reduced side-effects and, most importantly, the first uterine pass effect.

Biological Availability↗
PubMed2000

Roles of KIT and KIT LIGAND in ovarian function.

Evidence from mouse mutants indicates that the Kit gene encoding KIT, a receptor present on the oocyte and theca cells, and the Mgf gene encoding KIT LIGAND, the ligand of KIT, are important regulators of oogenesis and folliculogenesis. Recently, in vitro cultures of fetal gonads, of follicles and of oocytes have identified specific targets for the KIT-KIT LIGAND interaction. In fetal gonads, an anti-apoptotic effect of KIT-KIT LIGAND interactions on primordial germ cells, oogonia and oocytes has been demonstrated. In postnatal ovaries, the initiation of follicular growth from the primordial pool and progression beyond the primary follicle stage appear to involve KIT-KIT LIGAND interactions. During early folliculogenesis, KIT together with KIT LIGAND controls oocyte growth and theca cell differentiation, and protects preantral follicles from apoptosis. Formation of an antral cavity requires a functional KIT-KIT LIGAND system. In large antral follicles, the KIT-KIT LIGAND interaction modulates the ability of the oocyte to undergo cytoplasmic maturation and helps to maximize thecal androgen output. Hence, many steps of oogenesis and folliculogenesis appear to be, at least in part, controlled by paracrine interactions between these two proteins.

Animals↗
PubMed1999

Dehydroepiandrosterone (DHEA) response to i.v. ACTH in patients with chronic fatigue syndrome.

Previous studies have demonstrated concentrating neuroendocrinological disturbances in chronic fatigue syndrome (CFS) patients, concentrating in particular on low cortisol levels and a hypothalamic deficiency. In order to investigate the dynamic response of the adrenal glands, we measured dehydroepiandrosterone (DHEA) in serum after adreno-corticotropic hormone (ACTH) stimulation during 60 minutes in 22 CFS-patients and 14 healthy controls. We found normal basal DHEA levels, but a blunted serum DHEA response curve to i.v. ACTH injection. This observation adds to the large amount of evidence of endocrinological abnormalities in CFS. Relative glucocorticoid deficiency might contribute to the overall clinical picture in CFS, and could explain some of the immunological disturbances observed in this syndrome.

Adrenal Glands↗
PubMed1999

Luteal phase and clinical outcome after human menopausal gonadotrophin/gonadotrophin releasing hormone antagonist treatment for ovarian stimulation in in-vitro fertilization/intracytoplasmic sperm injection cycles.

The luteal phase hormonal profile and the clinical outcome of 69 patients undergoing in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) after ovarian stimulation with human menopausal gonadotrophin (HMG) and the gonadotrophin-releasing hormone (GnRH) antagonist Cetrorelix were analysed. Twenty-four patients received Cetrorelix 0.5 mg (group I) while in 45 patients Cetrorelix 0.25 mg was administered (group II). Human chorionic gonadotrophin (HCG) was used as luteal support. Nine clinical pregnancies were obtained in group I (37.5%) and 12 in group II (26. 6%). These results were not significantly different. Serum progesterone and oestradiol concentrations did not differ between the two groups either in pregnant or non-pregnant patients. An expected decrease of the same hormones was observed 8 days after the pre-ovulatory HCG injection in non-pregnant women. With regard to serum luteinizing hormone concentrations, a decrease was observed 2 days after the pre-ovulatory HCG injection and was maintained at almost undetectable levels throughout the entire luteal phase in both conception and non-conception cycles of group I and group II. This study demonstrates that different doses of GnRH antagonist do not have any impact on the luteal phase of IVF/ICSI cycles when hormonal support is given.

Abortion, Spontaneous↗