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Biomedical subjects

Johan Smitz

Publications and source records attributed to Johan Smitz.

At least 19 recordsLinked to original sources
PubMed2007

Principal findings from a multicenter trial investigating the safety of follicular-fluid meiosis-activating sterol for in vitro maturation of human cumulus-enclosed oocytes.

OBJECTIVE: To evaluate the safety of applying follicular-fluid meiosis-activating sterol (FF-MAS) in vitro to immature human oocytes. DESIGN: Phase I bicenter, randomized, parallel-group, controlled, partially blinded trial. SETTING: Third-level referral academic centers, including reproductive biology and genetics laboratories. PATIENTS: Endocrinologically normal women with a medical indication for IVF or intracytoplasmic sperm injection, or healthy volunteers. INTERVENTION(S): Subjects were randomized at a ratio 1 to 6 into either conventional GnRH-agonist and recombinant FSH stimulation (IVO) for oocyte retrieval, or minimally stimulated in vitro maturation (IVM) with the use of recombinant FSH. Retrieved immature oocyte cumulus complexes were cultured for 30 or 36 hours in one of six IVM culture conditions containing FF-MAS (range, 0.1-20 microM). Polar body-extruded oocytes from the IVO and IVM groups were processed for chromosomal analysis. MAIN OUTCOME MEASURE(S): The primary endpoint was the incidence of metaphase II stage oocytes with numeric chromosomal abnormalities, using full (spectral karyotyping) or partial (fluorescent in situ hybridization with seven probes) karyotyping or Giemsa count. A secondary objective was to document the frequency of metaphase II oocytes after IVM with FF-MAS supplements. RESULT(S): Oocyte cumulus complexes obtained from the IVO (mean, 8.9) and IVM (mean, 6.2) groups had equal maturation rates. Compared to IVO, exposure of germinal-vesicle oocytes for a maturation period of 30 hours did not increase aneuploidy. An exposure period of 36 hours doubled the aneuploidy rate, but this was significant only for the 20-muM dose of FF-MAS. CONCLUSION: Inclusion of 1-10 microM FF-MAS in a 30-hour IVM protocol is safe.

Adult↗
PubMed2006

Spuriously elevated serum estradiol concentrations measured by an automated immunoassay rarely cause unnecessary cancellation of in vitro fertilization cycles.

OBJECTIVE: To report a case of a patient in whom serum E2, as measured by an automated E2 assay, remained elevated at levels corresponding to the periovulatory phase (>380 pg/mL), despite 3 weeks of treatment by a GnRH agonist (GnRH-a). DESIGN: Case report. SETTING: Tertiary-care academic center. PATIENT(S): A 39-year-old patient accepted for IVF treatment. In view of controlled ovarian hyperstimulation, down-regulation by a GnRH-a was monitored by serum E2 measurement. INTERVENTION(S): Obvious causes for high serum E2 levels (pregnancy, E2-producing ovarian cysts, and noncompliance of the patient) were excluded. The IVF cycle was canceled. MAIN OUTCOME MEASURE(S): Serum samples were analyzed retrospectively by radioimmunoassay (RIA), and ether extraction of the samples was performed before measurement by the automated E2 assay. RESULT(S): Radioimmunoassay revealed adequately suppressed serum E2 levels under GnRH-a treatment (<15 pg/mL). Ether extraction revealed an interfering component that was soluble in the aqueous phase. CONCLUSION(S): If a patient on a long controlled ovarian hyperstimulation IVF protocol still has high serum E2 levels after 2 to 3 weeks of down-regulation, and obvious reasons for this (pregnancy, E2 producing ovarian cysts, noncompliance of the patient) are excluded, E2 immunoassay interference should be excluded to avoid unnecessary cancellation of the IVF cycle.

Adult↗
PubMed2006

Oocyte maturity and preimplantation development in relation to follicle diameter in gonadotropin-releasing hormone agonist or antagonist treatments.

OBJECTIVE: To analyze the distribution of oocytes nuclear maturational stages and intracytoplasmic sperm injection (ICSI) outcome in relation to follicular size, after controlled ovarian hyperstimulation (COH), using gonadotropin-releasing hormone (GnRH) antagonist or agonist protocols. DESIGN: Prospective comparative study. SETTING: Fertility unit in an academic hospital. PATIENT(S): Normo-ovulatory patients with unexplained, male and/or tubal factor infertility undergoing ICSI treatment. INTERVENTION(S): Oocytes were retrieved from small (7-12 mm) and large follicles (>12 mm). MAIN OUTCOME MEASURE(S): Oocyte nuclear maturation stage, fertilization and pre-implantation embryonic development of the mature ova evaluated. RESULT(S): Distribution of oocyte maturation stages from small follicles did not differ between the two COH protocols. From large follicles, a statistically significantly higher percentage of immature oocytes was retrieved after antagonist compared with agonist treatment (16 vs 11%, respectively). No statistically significant differences existed regarding ICSI outcome of mature ova within the same follicle diameter range between COH protocols. Inseminated ova from small follicles presented a significantly higher number of embryos with delayed development on day 3 after ICSI. CONCLUSION(S): A greater heterogeneity of maturity was found in the population of oocyte stages that were present after the antagonist protocol. Matured oocytes retrieved from small follicles generated embryos of lower developmental potential than oocytes derived from larger follicles.

Adult↗
PubMed2006

Morphological and ultrastructural evaluation of cultured frozen-thawed human fetal ovarian tissue.

OBJECTIVE: To investigate a defined culture condition for the culture of frozen-thawed human ovarian tissue. DESIGN: Prospective laboratory study. SETTING: Reproductive biology laboratories in university hospitals. PATIENT(S): Fetal ovarian tissue from elective termination of pregnancy. INTERVENTION(S): Culture of frozen-thawed fetal ovarian tissue for up to 63 days. MAIN OUTCOME MEASURE(S): Morphology, morphometry, and survival of follicles in relation to culture times. RESULT(S): The proportion of primordial, early primary, and primary follicles in frozen-thawed (day 0) ovarian tissue was 77.5%, 21.7%, and 0.8%, respectively. Pronounced degeneration was found in all cell types, and < or =36% of the follicles had signs of atresia at days 7-14, but this figure improved with culture time to <20% of the total follicular population. After 7-14 and 21-35 days of culture, the relative proportion of the follicles in the different classes remained nearly stable. Morphometric examination of healthy follicles showed a significant increase in both follicle and oocyte diameter compared with control. A few follicles had developed to the early secondary stage. Ultrastructural analysis demonstrated well-preserved morphological integrity of healthy primordial and early primary follicles. Immunohistochemical localization of proliferating cell nuclear antigen was positive in proliferating follicular cells at days 7-14 and 21-35 of culture. CONCLUSION(S): The present culture condition leads to good survival and progressive follicular growth and differentiation that is comparable to the physiological pattern of early folliculogenesis.

Aborted Fetus↗
PubMed2006

Aromatase inhibitors in ovarian stimulation for IVF/ICSI: a pilot study.

This prospective randomized pilot study was aimed at investigating the effect of the novel addition of aromatase inhibitors to an ovarian stimulation protocol for IVF or intracytoplasmic sperm injection, on endocrine parameters including serum androgen, oestrogen, progesterone, LH and FSH concentrations. The patients were randomized to receiving letrozole (group A; n = 10), versus no letrozole (group B; n = 10) in an ovarian stimulation protocol with recombinant FSH 150 IU/day starting on day 2 of the cycle, and gonadotrophin-releasing hormone antagonist 0.25 mg/day starting on day 6 of the cycle. Median LH concentrations were significantly higher (P < 0.01) in group A versus group B during letrozole administration. Median serum oestradiol concentrations were lower in group A versus group B, and median serum FSH, testosterone and androstenedione concentrations were higher in group A versus group B, throughout the follicular phase, without reaching significance. Median endometrial thickness was significantly higher (P < 0.05) in group A versus group B on the day of human chorionic gonadotrophin administration. Pregnancies were achieved. This pilot study supports the idea that aromatase inhibitors can contribute to normal potential of implantation and follicular response, without having negative anti-oestrogenic effects.

Adult↗
PubMed2006

Alpha-fetoprotein controls female fertility and prenatal development of the gonadotropin-releasing hormone pathway through an antiestrogenic action.

It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.

Androstatrienes↗
PubMed2005

Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole.

Aneuploidy tests are important in evaluating genetic hazards especially when chemical exposures are suspected to affect the fidelity of chromosome segregation in oocytes and embryos. In the current study, a newly established method, mouse preantral follicle culture, was employed to grow oocytes in vitro within follicles. The sensitivity of in vitro grown follicle enclosed oocytes was compared with oocytes maturing in vivo in the ovary. In both the cases, oocytes were exposed to the cytostatic chemical, nocodazole, from the time of hormonally stimulated resumption of meiosis. The in vivo study revealed a significant decrease in the number of ovulated mouse oocytes and an increase in meiosis I-arrested and hyperploid metaphase II oocytes at a single i.p. dose of 70 mg/kg body weight of nocodazole. A significant increase was also observed in the number of meiosis I-arrested and hyperploid mouse oocytes from preantral follicle culture, when they were cultured in the presence of >or=30 nM nocodazole during the final stages of maturation. This concentration is slightly lower than that previously shown to induce nondisjunction in denuded mouse oocytes or in cultured human lymphocytes. The higher sensitivity of the in vitro matured oocytes from preantral follicle culture than that of denuded oocytes may be related to a synergistic adverse influence of nocodazole on the oocyte, on somatic cell integrity and on cell-cell communication, which possibly also affects ovulation in vivo. When expressed in molarity relative to the mouse weight, the effective dose of the acute exposure in vivo is 3-4 orders of magnitude higher than the lowest effective concentration employed continuously in vitro. Reduced bioavailability of nocodazole to the target cells due to its poor water solubility may contribute to this difference. Preantral follicle culture can be helpful in analysing mechanisms in chemically induced aneuploidy in mammalian oogenesis, and in predicting the consequences of chemical exposures in vivo.

Aneuploidy↗
PubMed2005

Serum hormones for predicting pregnancy outcome after assisted reproductive technology.

Serum human chorionic gonadotrophin (HCG) in the second and third week after embryo transfer has been used for prediction of pregnancy outcome after assisted reproduction. There are few data on the clinical utility of HCG, progesterone and oestradiol, measured by contemporary immunoassay, in the fourth week after embryo transfer and later. Moreover, large inter-method differences have been described between automated immunoassays, making method-specific cut-off values mandatory. The main aim of this study was to determine assay-specific optimal cut-off values for serum HCG, progesterone and oestradiol for prediction of clinical pregnancy outcome in singleton pregnancies after assisted reproductive techniques, at days 11, 18 and 25 and at week 6 after embryo transfer. A retrospective study was performed on frozen serum samples of 67 singleton pregnancies after assisted reproduction techniques. HCG, oestradiol and progesterone were determined with the automated (random access) VIDAS immunoanalyser. Receiver operating characteristic curve analysis was performed to determine optimal cut-off values. Predictive values were calculated based on the prevalence of non-viable pregnancy after assisted reproduction. It was concluded that measurement of HCG by VIDAS at days 18 and 25, and at week 6 after embryo transfer yields high positive (70.5-100%) and negative (87.2-94.4%) predictive values for clinical pregnancy outcome.

Chorionic Gonadotropin↗
PubMed2005

Evaluation of a new automated electrochemiluminescent sex hormone-binding globulin (SHBG) immunoassay.

Serum sex hormone-binding globulin (SHBG) regulates the cellular bioavailability of SHBG-bound steroid hormones. Since variations in SHBG levels may affect the concentration of free, i.e., biologically active testosterone in serum, SHBG levels are commonly measured as a supplement to total testosterone determination. The recently developed electrochemiluminescence Elecsys SHBG immunoassay was evaluated analytically on a Modular E170 (Roche Diagnostics, Mannheim, Germany) immunoanalyzer. Major differences in SHBG concentrations have been described among the commercially available methods; we therefore compared the new method with an established SHBG immunoradiometric assay (IRMA) in 99 routine serum samples. To provide reference values to clinicians, SHBG concentration was measured by Elecsys in 304 serum samples from healthy volunteers and several relevant clinical subgroups. The within-run and total imprecision coefficients of variation were </=2.9% and </=3.3%, respectively. Functional sensitivity was at least 0.74 nmol/L. Recoveries after dilution of high-concentration samples in low-titer human serum or in assay diluent were within the range of 85-110%. The Elecsys SHBG assay correlated well (r=0.98) with the SHBG immunoradiometric assay, but values were higher for the Elecsys assay (Passing Bablok regression analysis: slope 1.14, intercept +2.5). In healthy subjects and clinical subgroups, we confirmed the differences in SHBG values reported in the literature. The Elecsys SHBG immunoassay provides precision and reliability in combination with reduced turnaround time.

Contraceptives, Oral, Hormonal↗
PubMed2004

A reproducible two-step culture system for isolated primary mouse ovarian follicles as single functional units.

A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 microm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of alpha-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 microm were transferred to alpha-MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days. Overall, more than 85% of the follicles survived the first culture step, and oocyte growth and granulosa cell proliferation had increased by 25% (P < 0.05). Follicle survival at Day 18 was related to initial follicle diameters at isolation. Average meiotic maturation rates and estrogen secretion were lower compared to those of cultures starting with early preantral follicles of 100-130 microm. Although reverse transcription-polymerase chain reaction analysis revealed the presence of LH-receptor mRNA in thecal cells, an exogenous androstenedione replacement resulted in an increase of estrogen production, suggesting substrate insufficiency. The time needed to grow from early preantral stages to in vitro ovulation is strongly dependent on the initial follicle diameter at isolation. Morphological characteristics of cultured follicles were suggestive for combined transforming growth factor beta deficiencies during in vitro culture.

Animals↗
PubMed2004

Number of ovarian follicles in human fetuses with the 45,X karyotype.

OBJECTIVE: To evaluate the numbers of ovarian follicles during fetal life in the gonads of human female fetuses with the 45,X karyotype (Turner syndrome, TS) and to compare them with those from age-matched 46,XX fetuses. DESIGN: Retrospective study. SETTING: An academic hospital. PATIENT(S): Ovarian samples from TS fetuses (aged 17-37 weeks), mainly obtained after induced abortion, and ovaries from eight control fetuses (matching gestational ages). INTERVENTION(S): Embedded blocks of ovaries were collected from anatomy-pathology departments of three university hospitals and were sectioned and stained with hematoxylin-eosin. MAIN OUTCOME MEASURE(S): Observation of primordial and growing follicles. RESULT(S): In the fetal ovaries of controls, numerous oogonia were observed at 18 weeks. Primordial follicles were present in all ovaries from 20 weeks' gestation onward, whereas preantral and antral follicles were observed from 26 weeks onwards. In ovaries from 45,X TS fetuses, oogonia were observed in some ovaries, but no primordial, preantral, or antral follicles were found, even in ovaries from the third trimester of gestation. CONCLUSION(S): Follicle formation and growth are severely reduced in ovaries from aborted 45,X TS fetuses.

Abortion, Induced↗
PubMed2004

Administration of gonadotropin-releasing hormone antagonist from day 1 of stimulation in in vitro fertilization.

After administration of GnRH antagonist from day 1 of stimulation for IVF, 12.1 cumulus oocyte complexes were retrieved (95% confidence interval [CI], 10.6-13.6), 1.9 embryos were transferred (95% CI, 1.7-2.0), and 2.4 embryos were cryopreserved (95% CI, 1.7-3.1). Stimulation for 8.8 days (95% CI, 8.4-9.2) with recombinant FSH resulted in an ongoing implantation rate of 26.5% (95% CI, 17.1-35.9) and an ongoing pregnancy rate of 39.7% per started cycle (95% CI, 30.1-50.8) and of 42.4% per ET (95% CI, 32.3-53.1).

Adult↗
PubMed2004

Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.

The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis. Isolated mouse oocytes resuming meiosis and progressing to metaphase II in vitro have recently been used to assess targets, aneugenic potential and sensitivity of oocytes to chemical exposures. In order to extend in vitro maturation tests to earlier stages of oogenesis, an in vitro assay with mouse preantral follicle cultures has been established. It permits the identification of direct and also indirect effects of environmental chemicals on the somatic compartment, the follicle and theca cells, that may lead to disturbances of oocyte growth, maturation and chromosome segregation. Early preantral follicles from prepubertal female mice are cultured in microdroplets for 12 days under strictly controlled conditions. The follicle-enclosed oocytes resume maturation, develop to metaphase II and become in vitro ovulated within 16 h after a physiological ovulatory stimulus with recombinant human gonadotrophins and epidermal growth factor. These oocytes grown and matured in vitro possess normal barrel-shaped spindles with well-aligned chromosomes. Their chromosomes segregate with high fidelity during anaphase I. The model aneugen colchicine induced a meiotic arrest and aneuploidy in these in vitro grown, follicle-enclosed oocytes in a dose-dependent manner, comparable to in vivo tests. Therefore, preantral follicle culture appears to provide an effective and reliable method to assess the influences of environmental mutagens, pharmaceutical agents and potentially endocrine disrupting chemicals on the fidelity of female meiosis.

Aneugens↗
PubMed2004

Multicentre performance evaluation of the E170 module for modular analytics.

The E170 module was evaluated at 13 sites in an international multicentre study. The objective of the study was to assess the analytical performance of 49 analytes, and to collect feedback on the system's reliability and practicability. The typical, within-run coefficients of variation (CVs) for most of the quantitative assays ranged between 1 and 2% while a range of 2-4% was achieved with the infectious disease methods. Total precision CVs were found to be within the manufacturer's expected performance ranges, demonstrating good concordance of the system's measuring channels and a high reproducibility during the 2-4-week trial period. The functional sensitivity of 11 selected assays met the clinical requirements (e.g., thyreotroponin (TSH) 0.008 mU/l, troponin T 0.02 microg/l, total prostate-specific antigen (PSA) 0.03 microg/l). The E170 showed no drift during an 8-hour period and no relevant reagent carryover. Accuracy was confirmed by ring trial experiments and method comparisons vs. Elecsys 2010. The reliability and practicability of the system's hardware and software met with, or even exceeded, the evaluator's requirements. Workflow studies showed that E170 can cover the combined workload of various routine analysers in a variety of laboratory environment. Throughput and sample processing time requirements were achieved while personnel 'hands-on-time' could be reduced.

Calibration↗