Influence de deux milieux séquentiels pour la culture d'embryons sur la production d'hormone gonadotrophine chorionique et de progestérone par des cellules JEG-3 et BeWo [Effects of different embryo development media on hCG and progesterone release by JEG-3 and BeWo cells]

Current in vitro fertilisation (IVF) practice requires synchronisation between the¦environment of cultured oocytes and embryos and the surroundings to what they would have¦been exposed to in vivo. Commercial, sequential media follow this requirement but their exact¦composition is not available. We have compared two widely used IVF culture media systems using¦the two choriocarcinoma cell lines JEG-3 and BeWo. The two hormones hCG and progesterone¦were determined in the culture supernatants as endpoints. In both cell lines, but in a more¦pronounced way in JEG-3, progesterone rather than hCG production was stimulated, and a¦higher hormone release was observed in the fertilisation than in the cleavage media. Differences¦between manufacturers were small and did not favour one system over the other. We conclude¦that both sequential media systems can be equally well used in current IVF laboratory practice.¦© 2012 Elsevier Masson SAS. All rights reserved.

Randomized controlled trial comparing highly purified (HP-hCG) and recombinant hCG (r-hCG) for triggering ovulation in ART.

BACKGROUND: A randomized controlled trial (RCT) comparing highly purified human Choriogonadotrophin (HP-hCG) and recombinant hCG (r-hCG) both administered subcutaneously for triggering ovulation in controlled ovarian stimulation (COS) for Assisted Reproductive Technology (ART). METHODS: Multi-centre (n = 4), prospective, controlled, randomized, non-inferiority, parallel group, investigator blind design, including 147 patients. The trial was registered with www.clinicaltrials.gov, using the identifier: NCT00335569. The primary endpoint is the number of oocytes retrieved, while the secondary endpoints include embryo implantation, pregnancy and delivery rates as well as safety parameters. RESULTS: The number of retrieved oocytes was not inferior when HP-hCG was used as compared to r-hCG: the mean number was 13.3 (6.8) in HP-hCG and 12.5 (5.8) in the r-hCG group (p = 0.49) with a 95% CI (-1.34, 2.77). Regarding the secondary outcomes, there were also no differences in fertilization rate at 57.3% (467/815) vs. 61.3% (482/787) (p = 0.11), the number of embryos available for transfer and cryopreservation (2PN stage) and implantation, pregnancy and delivery rates. Furthermore, there were no differences in the number and type of adverse events reported. HP-hCG was therefore not inferior to r-hCG. CONCLUSIONS: HP-hCG and r-hCG are equally efficient and safe for triggering ovulation in ART and, both being administered subcutaneously, equally practical and well tolerated by patients.

Influences of β-HCG administration on carbon isotope ratios of endogenous urinary steroids.

Several factors influencing the carbon isotope ratios (CIR) of endogenous urinary steroids have been identified in recent years. One of these should be the metabolism of steroids inside the body involving numerous different enzymes. A detailed look at this metabolism taking into account differences found between steroids excreted as glucuronides or as sulphates and hydrogen isotope ratios of different steroids pointed out possibility of unequal CIR at the main production sites inside the male body - the testes and the adrenal glands. By administration of β-HCG it is possible to strongly stimulate the steroid production within the testes without influencing the production at the adrenal glands. Therefore, this treatment should result in changed CIR of urinary androgens in contrast to the undisturbed pre-treatment values. Four male volunteers received three injections of β-HCG over a time course of 5 days and collected their urine samples at defined intervals after the last administration. Those samples showing the largest response in contrast to the pre-administration urines were identified by steroid profile measurements and subsequent analysed by GC/C/IRMS. CIR of androsterone, etiocholanolone, testosterone, 5α- and 5β-androstanediol and pregnanediol were compared. While pregnanediol was not influenced, most of the investigated androgens showed depleted values after treatment. The majority of differences were found to be statistically significant and nearly all showed the expected trend towards more depleted δ(13)C-values. These results support the hypothesis of different CIR at different production sites inside the human body. The impact of these findings on doping control analysis will be discussed.

Influence of multiple injections of human chorionic gonadotropin (hCG) on urine and serum endogenous steroids concentrations.

Since it is established that human chorionic gonadotropin (hCG) affects testosterone production and release in the human body, the use of this hormone as a performance enhancing drug has been prohibited by the World Anti-Doping Agency. Nowadays, the only validated biomarker of a hCG doping is its direct quantification in urine. However, this specific parameter is subjected to large inter-individual variability and its determination is directly dependent on the reliability of hCG immunoassays used. In order to counteract these weaknesses, new biomarkers need to be evidenced. To address this issue, a pilot clinical study was performed on 10 volunteers submitted to 3 subsequent hCG injections. Blood and urine samples were collected during two weeks in order to follow the physiological effects on related compounds such as the steroid profile or hormones involved in the hypothalamo-pituitary axis. The hCG pharmacokinetic observed in all subjects was, as expected, prone to important inter-individual variations. Using ROC plots, level of testosterone and testosterone on luteinizing hormone ratio in both blood and urine were found to be the most relevant biomarker of a hCG abuse, regardless of inter-individual variations. In conclusion, this study showed the crucial importance of reliable quantification methods to assess low differences in hormonal patterns. In regard to these results and to anti-doping requirements and constraints, blood together with urine matrix should be included in the anti-doping testing program. Together with a longitudinal follow-up approach it could constitute a new strategy to detect a hCG abuse, applicable to further forms of steroid or other forbidden drug manipulation.

Serum-Mediane von Placentaprotein-Markern: Relevanz der Unterschiede zwischen spontanen und nach-in-vitro-Fertilisation eingetretenen Schwangerschaften für das fetale Trisomie-Screening.

Trisomy-21 (Down syndrome) is the most frequent chromosomal abnorm- ality but only one third of cases would be detected by amniocentesis based on maternal age alone. Serum screening tests in the early second trimester increase the detec- tion rate to 60-65%, and more recently it was found that such screening was also possible in the first trimester by quantifying a diffe- rent panel of markers. The concen- trations of these placental proteins are strongly dependent on gestatio- nal age; thus control medians must be established and precise dating is essential. Serum chorionic gonado- trophin (HCG) levels were recently found to be increased in IVF preg- nancies compared to spontaneous gestations, leading to a falsely ele- vated trisomy screening risk. The aim of this preliminary study was to find out whether, in the first-trime- ster screening, the markers similarly differed between IVF and spontane- ous pregnancies which would call for the establishment of separate normal medians for IVF patients. We compared 24 pregnancies ob- tained after ovarian stimulation and IVF with six women after thawed embryo transfer (unstimulated cycle) and 63 gestation- and maternal-age matched spontaneously pregnant controls. A single serum was ob- tained between 6 and 16 weeks of gestation and various placental protein levels determined by im- munometric assays. Serum levels of pregnancy-associated plasma protein A (PAPP-A), which is the major marker in the first-trimes- ter screening test, were reduced in IVF pregnancies: after 9 weeks of gestation, multiples of median (MoMs) ranged between 0.23 and 3.58 (logarithmic mean 0.743). For the frozen/thawed transfers, this value was 1.08. In the 9-12 week group containing 6 cases of IVF, three thawed transfers and 25 con- trols, PAPP-A was significantly redu- ced in the stimulated compared to the nonstimulated cycles. In the late first and early second trimester the difference was not significant in our small group but the trend persisted. Pregnancies after IVF will thus show an increased incidence of false positive results in fetal trisomy-21 screening, and special medians should be established for these pati- ents.

Evaluation of two immunoassays for the measurement of human chorionic gonadotropin in urine for anti-doping purposes.

Background: Urinary human chorionic gonadotropin (hCG) concentration is routinely measured in all anti-doping laboratories to exclude the misuse of recombinant or urinary hCG preparations. In this study, extended validation of two commercial immunoassays for hCG measurements in urine was performed. Both tests were initially designed for hCG determination in human serum/plasma. Methods: Access (R) and Elecsys (R) 1010 are two automated immunoanalysers for central laboratories. The limits of detection and quantification, as well as intra-laboratory and inter-technique correlation, precision, and accuracy, were determined. Stability studies of hCG in urine following freezing and thawing cycles (n = 3) as well as storage conditions at room temperature, 4 degrees C and 20 degrees C, were performed. Results: Statistical evaluation of hCG concentrations in male urine samples (n = 2429) measured with the Elecsys (R) 1010 system enabled us to draw a skewed frequency histogram and establish a far outside value equal to 2.3 IU/L. This decision limit corresponds to the concentration at which a sportsman will be considered positive for hCG. Intra-assay precision for the Access (R) analyser was less than 4.0 A, whereas the inter-assay precision was closer to 4.5 % (concentrations of the official external controls contained between 5.5 and 195.0 IU/L). Intra and inter-assay precision for the Elecsys (R) 1010 analyser was slightly better. A good inter-technique correlation was obtained when measuring various urine samples (male and female). No urinary hCG loss was observed after two freeze/thaw cycles. On the other hand, time and inappropriate storage conditions, such as temperatures above 10 degrees C for more than 5 days, can deteriorate urinary hCG. Conclusions: Both analysers showed acceptable performances and are suitable for screening urine for anti-doping analyses. Each laboratory should validate and establish its own reference values because hCG concentrations measured in urine can be different from one immunoassay to another. The time delay between urine collection and analysis should be reduced as much as possible, and urine samples should be transported in optimal conditions to avoid a loss of hCG immunoreactivity.

Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P < 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P < 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

Interstitial pregnancies' diagnosis and management: an eleven cases series.

PRINCIPLES: Interstitial pregnancy represents 2% of ectopic pregnancies, but it is a highly morbid condition with a 2.5% of maternal mortality. Its diagnostic and therapeutic management remains controversial. The aim of this review is to describe the management of interstitial pregnancy in our institution between 2001 and 2011 and to define some general rules for the clinical practice. METHODS: Single institution retrospective study. RESULTS: Eleven women were treated for interstitial pregnancy. The median age was 33 years and the median gestity was 4. Seven patients had a history of gynaecological surgery and four interstitial pregnancies followed in vitro fertilisation. The diagnosis was made at a median gestational age of seven weeks with a median beta-HCG level of 5,838 U/l. Six of the eleven patients received an initial treatment with intracornual methotrexate, three with intramuscular methotrexate and two with surgery. The median time to beta-HCG resolution was 58 days. Three of the eleven patients needed a second line treatment: two after intramuscular methotrexate and one after intracornual methotrexate. Six patients had further pregnancies and delivered by caesarean section. CONCLUSIONS: A high prevalence of previous ectopic pregnancies, gynaecological surgery and of pregnancies resulting from in vitro fertilisation was observed. The earliness of the diagnosis was the factor that allowed a conservative treatment in most cases. Beta-HCG level follow up was fundamental in allowing a second line therapy but beta-HCG can persist over a long period of time and this must be taken into account due to its possible psychological impact. Intracornual methotrexate seems to be more efficacious than intramuscular methotrexate in our series.

Prise en charge des séminomes sécrétants de l'hormone chorionique gonadotrope [Management of chorionic gonadotropin-producing seminoma.]

INTRODUCTION: The human chorionic gonadotropin (HCG)-producing seminoma is an uncommon entity and belongs to the overall category of pure seminoma. METHOD: The literature search was conducted on Medline(®) using the words: seminoma, human chorionic gonadotropin, HCG combined with radiotherapy, chemotherapy, surveillance, management and prognosis. We extended our search of similar references by related articles function, reading the bibliography of identified articles and publications available on Medline(®) from the same authors. This research was limited to English or French publications. Articles were eligible if they were randomized trials, prospective, retrospective or systematic reviews of the literature. RESULTS: Few articles were found on this subject. We selected the most relevant series while summarizing various parameters (epidemiological, clinical, therapeutic and prognostic). CONCLUSIONS: Clinical presentation, behaviour and work-up for HCG-producing seminoma should be the same as for non-secreting seminoma. HCG-producing seminoma tumours are not more resistant to radiation therapy or chemotherapy than non-secreting seminoma tumours. Radiotherapy remains an excellent option in stage I and IIA disease with chemotherapy as an alternative; overall prognosis is excellent. Surveillance in early stage HCG-producing seminoma is followed by a higher relapse than in early stage non-secreting seminoma.

Human chorionic gonadotropin in pregnancy and maternal risk of breast cancer.

Full-term pregnancies are associated with long-term reductions in maternal risk of breast cancer, but the biological determinants of the protection are unknown. Experimental observations suggest that human chorionic gonadotropin (hCG), a major hormone of pregnancy, could play a role in this association. A case-control study (242 cases and 450 controls) nested within the Northern Sweden Maternity Cohort included women who had donated a blood sample during the first trimester of a first full-term pregnancy. Total hCG was determined on Immulite 2000 analyzer. Odds ratios (OR) and 95% confidence intervals (CI) were estimated through conditional logistic regression. Maternal breast cancer risk decreased with increasing hCG (upper tertile OR, 0.67; CI, 0.46-0.99), especially for pregnancies before age 25 (upper tertile OR, 0.41; CI, 0.21-0.80). The association diverged according to age at diagnosis: risk was reduced after age 40 (upper tertile OR, 0.60; CI, 0.39-0.91) and seemed to increase before age 40 (upper tertile OR, 1.78; CI, 0.72-4.38). Risk was reduced among those diagnosed 10 years or longer after blood draw (upper tertile OR, 0.60; CI, 0.40-0.90), but not so among those diagnosed within 10 years (upper tertile OR, 4.33; CI, 0.86-21.7). These observations suggest that the association between pregnancy hCG and subsequent maternal risk of breast cancer is modified by age at diagnosis. Although the hormone seems to be a determinant of the reduced risk around or after age 50, it might not confer protection against, or it could even increase the risk of, cancers diagnosed in the years immediately following pregnancy.

Free β-human chorionic gonadotropin, total human chorionic gonadotropin and maternal risk of breast cancer.

BACKGROUND: We investigated whether the free β-human chorionic gonadotropin (free β-hCG) would provide additional information to that provided by total hCG alone and thus be useful in future epidemiological studies relating hCG to maternal breast cancer risk. MATERIALS & METHODS: Cases (n = 159) and controls (n = 286) were a subset of our previous study within the Northern Sweden Maternity Cohort on total hCG during primiparous pregnancy and breast cancer risk. RESULTS: The associations between total hCG (hazard ratio: 0.79; 95% CI: 0.49-1.27), free β-hCG (hazard ratio: 0.85; 95% CI: 0.33-2.18) and maternal risk of breast cancer were very similar in all analyses and mutual adjustment for either one had minor effects on the risk estimates. CONCLUSION: In the absence of a reliable assay on intact hCG, total hCG alone can be used in epidemiological studies investigating hCG and breast cancer risk, as free β-hCG does not appear to provide any additional information.

Dose and time effects of treatment with low doses of a LRH agonist on testicular axis and accessory sex organs in rats.

LRH and its agonists have been shown to exert both stimulatory and inhibitory effects on testicular function. In the present study, the dose and length of treatment were tested to determine the appearance of the stimulatory and inhibitory effects of LRH agonist on testicular axis including the three levels. Two doses of an agonist of LRH, 40 and 100 ng/100 g body weight (buserelin, 'agonist'), were administered daily for 1 to 15 days to adult male rats. Control rats received the vehicle only. On day 1, 2, 4, 8 and 15 of treatment, the pituitary, testicular and peripheral levels (weight of accessory sex organs and androgen receptors in ventral prostate) were tested 6 h after the last injection. For the 15 days of treatment with both doses, a stimulatory effect of the 'agonist' was observed on LH and FSH release. A short exposure (1-2 days) to the low dose of the 'agonist' had a stimulatory effect on the density of LH/hCG testicular receptors (326 +/- 49 vs control 185 +/- 21 fmol/mg protein, mean +/- SEM), on the weights of seminal vesicles and ventral prostate and exposure to both doses led to high plasma testosterone levels (13.8 +/- 0.5 and 13.7 +/- 0.7 ng/ml, respectively, vs control 2.6 +/- 0.3 ng/ml), and to an increased density of nuclear androgen receptors in the ventral prostate (142 +/- 9 and 144 +/- 15 fmol/mg protein respectively vs control 97 +/- 12 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol levels in men with congenital hypogonadotropic hypogonadism and the effects of different modalities of hormonal treatment.

Objective: To evaluate the degree of E-2 deficiency in male congenital hypogonadotropic hypogonadism (CHH), and its response to different hormonal treatments.Design: Retrospective and prospective studies.Setting: Academic institution.Patient(s): Untreated or treated CHH, healthy men, untreated men with Klinefelter syndrome (KS). Intervention(s): Serum sex hormone-binding globulin (SHBG) and total E-2 (TE2) as well as bioavailable (BE2) and free (FE2) levels were measured and determined.Main Outcome Measure(s): Total, bioavailable, and free testosterone, TE2, BE2, FE2 were compared in normal men, untreated and treated CHH and in untreated KS.Result(s): TE2, BE2, and FE2 levels were very significantly lower in untreated patients with CHH (n = 91) than in controls (n = 63) and in patients with KS (n = 45). The TE2 correlated positively with serum total T in patients with CHH. The TE2 also correlated very positively with serum LH in the combined population of patients with CHH and healthy men, suggesting that low E-2 levels in CHH are due to severe LH-driven T deficiency. All fractions of circulating E-2 were very significantly higher in patients with CHH receiving T enanthate (n = 101) or the FSH-hCG combination (n = 88) than in untreated patients with CHH. Contrary to dihydrotestosterone (DHT), both T enanthate and combined FSH-hCGtherapy significantly and prospectively increased TE2 levels in patients with CHH.Conclusion(s): Contrary to KS, the male hypogonadism observed in CHH is associated with profound E-2 deficiency, which can be overcome by aromatizable androgen or combined gonadotropin therapy. (Fertil Steril (R) 2011; 95: 2324-29. (C) 2011 by American Society for Reproductive Medicine.)