Oocyte maturity, oocyte fertilization and cleavage-stage embryo morphology are better in natural compared with high-dose gonadotrophin stimulated IVF cycles.

RESEARCH QUESTION: Does high-dose gonadotrophin stimulation have an effect on oocyte and early-stage embryo development? DESIGN: This was a retrospective study including 616 natural cycle IVF (NC-IVF) and 167 conventional IVF (cIVF) cycles. In total, 2110 oocytes were retrieved and analysed in fresh cycles. In NC-IVF, only human chorionic gonadotrophin was applied to trigger ovulation. In cIVF, antagonist protocols with daily 150-300 IU of human menopausal gonadotrophins were performed. The effect of gonadotrophins on oocyte and early-stage embryo development was analysed. Primary outcomes were the occurrence of mature (metaphase II) oocytes, zygotes and embryos with good morphology at the cleavage stage 2 days after oocyte retrieval. RESULTS: The mature oocyte rate (number of mature oocytes/number of retrieved oocytes) was higher in NC-IVF than cIVF cycles (89% versus 82%, adjusted odds ratio [aOR] 1.79, P = 0.001), as was the zygote rate per oocyte retrieved (70% versus 58%, aOR 1.76, P = 0.001) and the zygote rate per mature oocyte (79% versus 71%, aOR 1.62, P = 0.001). The percentage of zygotes that developed into cleavage-stage embryos was no different. For the transferred embryos, the probability of having a good embryo morphology with four blastomeres and a fragmentation of <10% (score 0) in cleavage-stage embryos was found to be higher in NC-IVF (proportional aOR for four blastomeres 2.00, P < 0.001; aOR 1.87 for a fragmentation score of 0, P = 0.003). CONCLUSIONS: Oocyte maturity, oocyte fertilization and morphology of the cleavage-stage embryo are affected by high-dose gonadotrophin stimulation in fresh IVF cycles.

Gonadotrophin stimulation reduces follicular fluid hormone concentrations and disrupts their quantitative association with cumulus cell mRNA.

RESEARCH QUESTION: Do follicular fluid hormone concentrations and the mRNA expression of LHCG, FSH and androgen receptors, aromatase and anti-Müllerian hormone (AMH) in cumulus granulosa cells differ in naturally matured and stimulated follicles? DESIGN: Cross-sectional study involving 57 natural cycle IVF (NC-IVF) and 36 conventional gonadotrophin-stimulated IVF (cIVF) cycles performed between 2014 and 2016. cIVF was performed by ovarian stimulation with human menopausal gonadotrophin and gonadotrophin-releasing hormone antagonists. Hormone concentrations were determined in the follicular fluid of the leading follicle, and mRNA concentrations were quantified by reverse transcription polymerase chain reaction in RNA extracted from granulosa cells of the cumulus oophorus complex obtained from these fluids. RESULTS: Follicular fluid hormone concentrations were significantly lower in cIVF compared with NC-IVF follicles. Median concentrations were 0.50 and 14.5 mIU/ml for LH (P < 0.001), 16.1 and 46.5 nmol/l for testosterone (P < 0.001), 1270 and 2675 nmol/l for oestradiol (P < 0.001), and 12.3 and 28.9 pmol/l for AMH (P < 0.001), respectively. In cumulus granulosa cells, mRNA concentrations for LH receptor, FSH receptor, androgen receptor, aromatase and AMH did not differ between cIVF and NC-IVF follicles. Several hormone and mRNA concentrations were quantitatively associated in natural cycles such as follicular fluid AMH and cumulus granulosa cells AMH RNA (r2 = 0.107, P = 0.013), follicular fluid testosterone and cumulus granulosa cell AMH RNA (r2 = 0.158, P = 0.002), follicular fluid oestradiol and cumulus granulosa cell AMH RNA (r2 = 0.105, P = 0.014) and follicular fluid oestradiol and aromatase (r2 = 0.113, P = 0.011). In contrast, these associations were not observed in stimulated cycles. CONCLUSION: These findings indicate some effects of gonadotrophin stimulation on follicular physiology, which could be a cause for the previously suggested lower oocyte quality in cIVF compared with NC-IVF.

The endocrine milieu in naturally matured follicles is different in women with high serum anti-Müllerian hormone concentrations.

RESEARCH QUESTION: Is the endocrine milieu different in women with low serum anti-Müllerian hormone (AMH) concentration compared with women with high concentration? DESIGN: Cohort study of 84 women (four groups) classified according to AMH concentration and age undergoing natural cycle IVF treatment. Concentrations of LH, oestradiol, testosterone, androstenedione and AMH were determined in follicular fluid (FF), associations analysed and clinical outcome parameters evaluated. RESULTS: A positive correlation between serum and FF AMH concentrations was confirmed. Follicular fluid androstenedione concentration was positively correlated with serum AMH concentration (P < 0.0001, r2 = 0.197). The correlation between FF LH and FF testosterone concentration in all women was not significant (P = 0.050, r2 = 0.046); however, the correlation between FF androstenedione in women with high serum AMH concentration was significant (P = 0.032, r2 = 0.220). Follicular fluid testosterone and androstenedione were positively correlated with FF oestradiol overall and in some individual groups. The high serum AMH concentration group showed the highest FF AMH and androstenedione concentrations and lowest oestradiol-testosterone and oestradiol-androstenedione ratios. High FF AMH concentration was associated with a higher clinical pregnancy rate and high FF oestradiol concentration with a slightly better embryo quality. CONCLUSIONS: Differences in the endocrine milieu in women with high serum AMH concentration seem to be caused by increased follicular LH concentration. In women with high serum AMH concentration, FF androstenedione is increased and ratios of oestradiol-testosterone and oestradiol-androstenedione are decreased, suggesting a disturbed endocrine milieu caused by reduced metabolization of FF androgens into oestrogens. In natural cycles, FF AMH concentrations are positively associated with higher clinical pregnancy rates and oestradiol concentrations with a higher embryo score.

Gonadotropin Stimulation Has Only a Limited Effect on the Concentration of Follicular Fluid Signalling Proteins: An Antibody Array Analysis.

OBJECTIVE: The follicular fluid (FF) plays an essential role in the physiology of the follicle and the oocyte. Gonadotropin stimulation affects the FF steroid hormone and anti-Mullerian hormone (AMH) concentrations, which has been suggested to be the reason for lower oocyte competence in conventional gonadotropin stimulated in vitro fertilisation (cIVF) compared to natural cycle IVF (NC-IVF). To analyse the effect of gonadotropin stimulation on a broad spectrum of signalling proteins, we ran proteomic antibody arrays on FF of women undergoing both treatments NC-IVF and cIVF. METHOD: Twenty women underwent one NC-IVF and one cIVF treatment cycle. Follicular fluids of the first aspirated follicle were compared between the two groups using a protein microarray which included antibodies against 224 proteins related to cell signalling and reference proteins. Each of the 40 albumin-stripped, matched-pair samples was labelled in the reverse-dye (Cy3/Cy5) procedure before undergoing array hybridisation. Signal analysis was performed using normalisation algorithms in dedicated software. Five proteins yielding a value of P < 0.05 in the array experiment (Cystatin A, Caspase-3, GAD65/67, ERK-1, and ERK-2) were then submitted to quantitative determination by ELISA in the same follicular fluids. RESULTS: Array analysis yielded only a small number of differentially expressed signalling markers by unadjusted P values. Adjustment as a consequence of multiple determinations resulted in the absence of any significant differential marker expression on the array. Five unadjusted differentially expressed proteins were quantified immunometrically with antibodies from different sources. Follicular fluid concentrations of Cystatin A and MAP kinase ERK-1 concentrations were significantly higher in the cIVF than in the NC-IVF follicles, while GAD-2 (GAD65/67) did not differ. The assays for Caspase-3 and MAP kinase ERK-2 did not have the required sensitivities. CONCLUSION: In contrast to FF steroid hormones and AMH, FF concentrations of signalling proteins are not or only marginally altered by gonadotropin stimulation.

Serum and follicular fluid testosterone concentrations do not correlate, questioning the impact of androgen supplementation on the follicular endocrine milieu.

Basic research into a possible link between serum and follicular fluid androgen concentrations to detemine whether androgen supplementation in low responders affects follicular endocrine milieu is still lacking. Ninety-seven women (aged 28-43 years) undergoing one natural IVF cycle without any hormone stimulation were analysed. Serum and follicular fluid were collected at the time of follicle aspiration, and the concentrations of LH, total testosterone, oestradiol, dehydroepiandrosterone and anti-Mullerian hormone (AMH) were determined. Serum LH (P = 0.003) and AMH (P = 0.026) concentrations, and follicular fluid AMH (P = 0.015) decreased with increasing age. Within follicular fluid, total testosterone was correlated with oestradiol (P < 0.001) and AMH (P = 0.010); LH correlated with AMH (P = 0.005). Correlation analysis of serum and follicular fluid hormone concentrations revealed that LH, oestradiol and AMH correlated (P < 0.001), whereas testosterone did not. Testosterone serum concentrations did not correlate with other follicular fluid hormones, such as dehydroepiandrosterone, oestradiol and AMH, whereas serum LH correlated with follicular flulid AMH (P < 0.008). Follicular fluid hormone concentrations seem to be independent from serum testosterone. Therefore, it is questionable whether an increase in serum testosterone concentration by androgen supplementation could improve the follicular endocrine milieu.

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.