M. von Wolff

Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF

STUDY QUESTION Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are ∼3-fold higher, androstenedione (A2) is ∼1.5-fold higher and luteinizing hormone (LH) is ∼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'

Infertility is an increasing problem all over the world, and it has been estimated that 10–15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women’s health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The ‘Fruitful’ research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

Serum but not follicular fluid cytokine levels are increased in stimulated versus natural cycle IVF: a multiplexed assay study

Throughout follicular growth the number of immune cells increases, enhanced under stimulation with exogenous gonadotropins. This treatment, however, may adversely influence folliculogenesis and negatively affect oocyte quality through modifications in the follicular concentrations of cytokines released by these immune cells. We studied this hypothesis by systematically analysing the concentrations of cytokines present in the serum and follicular fluid at the time of follicular aspiration in conventional gonadotropin-stimulated (c-IVF) cycles in comparison with natural cycle IVF (NC-IVF) in which the follicles were naturally matured. Our study involved 37 NC-IVF and 39 c-IVF cycles including 13 women who underwent both therapies. Mean age was 35.3 ± 4.6 (SD) and 34.2 ± 3.7 years in the NC-IVF and c-IVF groups (ns). Thirteen cytokines were determined in matched serum and FF samples. Interleukin (IL)-4, TNF-α, RANTES, eotaxin and interferon-gamma-induced protein-10 concentrations were lower in FF than in serum. IL-6, -8, -10, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median levels in FF than in serum, indicating possible ovarian production. Most of these markers were also increased in concentration in the stimulated (c-IVF) than in the NC groups in the serum, but not in the follicular fluid. This finding can be attributed to the increased number of active follicles present after controlled ovarian stimulation. IL-8 was reduced in c-IVF cycles. Our study did not reveal differences in follicular fluid but in serum cytokine concentrations, suggesting that the follicular immune system might not be significantly affected by gonadotropin stimulation.

Intrafollicular inflammatory cytokines but not steroid hormone concentrations are increased in naturally matured follicles of women with proven endometriosis

PurposeThe aim of this study was to assess whether the intrafollicular cytokine profile in naturally developed follicles is different in women with endometriosis, possibly explaining the lower reproductive outcome in endometriosis patients.MethodsA matched case-control study was conducted at a university-based infertility and endometriosis centre. The study population included 17 patients with laparoscopically and histologically confirmed endometriosis (rAFS stages II–IV), each undergoing one natural cycle IVF (NC-IVF) treatment cycle between 2013 and 2015, and 17 age-matched NC-IVF women without diagnosed endometriosis (control group). Follicular fluid and serum was collected at the time of follicle aspiration. The concentrations of inflammatory cytokines (IL-1β, IL-6, IL-8, IL-15, IL-18, TNF-α) and hormones (testosterone, estradiol, AMH) were determined in follicular fluid and serum by single or multiplexed immunoassay and compared between both groups.ResultsIn the follicular fluid, IL-1β and IL-6 showed significantly (P < 0.001 and 0.01, respectively) higher median concentrations in the endometriosis group than in the control group and a tendency towards endometriosis severity (rAFS stage) dependence. The levels of the interleukins detectable in follicular fluid were significantly higher than those in the serum (P < 0.01). Follicular estradiol concentration was lower in severe endometriosis patients than in the control group (P = 0.036). Follicular fluid IL-1β and IL-6 levels were not correlated with estradiol in the same compartment in neither patient group.ConclusionsIn women with moderate and severe endometrioses, some intrafollicular inflammatory cytokines are upregulated and not correlated with intrafollicular hormone concentrations. This might be due to the inflammatory microenvironment in endometriosis women, affecting follicular function and thereby possibly contributing to the reproductive dysfunction in endometriosis.

Gonadotrophin stimulation in IVF alters the immune cell profile in follicular fluid and the cytokine concentrations in follicular fluid and serum

Study question Are the immune cell profiles and the cytokine concentrations in follicular fluid (FF) and serum at the preovulatory stage different in conventional exogenous gonadotrophin stimulated IVF (c-IVF) compared with natural cycle IVF (NC-IVF)? Summary answer The cell counts of CD45+ leucocytes and T cell subpopulations and the cytokine concentrations in FF and serum are different in c-IVF compared to NC-IVF. What is known already FF-derived cells are heterogeneous. Immune cells are involved in intra-ovarian processes and cytokines are required for normal follicular development. Gonadotrophins stimulate the regulatory intrafollicular system and influence the local distribution of immune cells and the intrafollicular release of cytokines. Administration of exogenous gonadotrophins may have a significant effect on this local regulatory system, which then in turn could influence oocyte quality. Study design, size, duration The study included 105 patients, 69 undergoing c-IVF and 36 undergoing NC-IVF. c-IVF was performed by exogenous ovarian stimulation with hMG and GnRH antagonists. Participants/materials, setting, methods FF samples were collected from the first dominant follicle in c-IVF without pooling and from single leading preovulatory follicles in NC-IVF. Three different approaches were used to analyze FF samples: (i) microscopic investigation of CD45+ leucocytes, (ii) fluorescence-activated cell sorting to determine CD19+ B cells and CD3+ T cells including T cell subpopulations (CD4+, CD8+), and (iii) evaluation of tumour necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), interleukins (IL)-2, -6, -8, -10 and vascular endothelial growth factor (VEGF) levels in matched FF and serum samples using the Bio-Plex® platform. Main results and the role of chance FF obtained from c-IVF contained proportionally more CD45+ leucocytes (P = 0.0384), but fewer CD8+ cytotoxic T cells than FF from NC-IVF. CD3+ T lymphocytes were the most common type of lymphocytes, and the number thereof was comparable in the two study groups. In c-IVF, serum VEGF levels were higher (P = 0.007) than in NC-IVF while FF contained marginally decreased concentrations of IL-8 in c-IVF in comparison to NC-IVF. The cytokine concentration gradient between FF and serum in c-IVF was 10-fold for IL-8 and 8-fold for VEGF and thereby markedly lower than in NC-IVF, where the differences were 32-fold and 30-fold, respectively. Strong positive correlations were determined between FF- IL-10 and FF- VEGF in c-IVF (r = 0.85, P < 0.0001) and in NC-IVF (r = 0.81, P < 0.0001). Large scale data N/A. Limitations, reasons for caution The ovulation of NC-IVF follicles was induced by the exogenous administration of hCG, which means that the environment did not fully correspond to the physiological situation. Wider implications of the findings The differences in the immune profile and the cytokine concentrations in c-IVF and NC-IVF follicles support the hypothesis that conventional ovarian stimulation affects indirectly and heterogeneously the intrafollicular milieu, and thereby possibly affects the oocyte quality and the IVF outcome. However, further studies are needed to confirm our findings and to refine stimulation protocols in the context of optimizing the intrafollicular environment during oocyte maturation. Study funding/competing interest(s) The study was supported by a research grant from IBSA Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. The authors are clinically involved in low dose mono-follicular stimulation and IVF-therapies, using gonadotrophins from all gonadotrophins distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH.

Generierung und Konservierung von Keimzellen

ZusammenfassungDie In-vitro-Maturation (IVM) ist eine Technik, welche die Reifung von Eizellen im Germinalvesikelstadium bis hin zur befruchtungsfähigen Metaphase-II-Eizelle beinhaltet. Die Eizellen werden in der Regel gezielt aus kleinen antralen Follikeln gewonnen. Der Erfolg nach 24- bis 36-stündiger Maturation wird derzeit mit der Bildung des 1. Polkörpers als Anzeichen für eine abgeschlossene erste meiotische Reifeteilung belegt. Die Qualität einer in vitro gereiften Eizelle kann durch ein neuartiges polarisationsmikroskopisches Verfahren noch besser eingeschätzt werden. Dieses Verfahren ermöglicht eine deutlich bessere Beurteilung und ist somit ein einzigartiges Instrument, um bestehende IVM-Protokolle zu optimieren. Zusammen mit der inzwischen realistischen Option der erfolgreichen Kryokonservierung von unreifen und reifen Eizellen durch die Vitrifikation eröffnen sich künftig für die IVM neue Einsatzgebiete.AbstractIn vitro maturation (IVM) is a technique which allows the maturation of oocytes from the germinal vesicle stage up to the stage of the fertilization-competent metaphase-II oocytes. Immature oocytes are primarily retrieved from small antral follicles. Their successful maturation is usually documented by formation of the first polar body as an indicator of completion of the first meiotic division. The quality of in vitro matured oocytes can now be judged by polarisation microscopy. This technique allows better quality assessment and is hence a unique instrument for optimising existing IVM protocols. In combination with the now realistic option of successful cryopreservation of mature and immature oocytes by the technique of vitrification, IVM will soon enter new fields of application.

Follicular flushing in natural cycle IVF does not affect the luteal phase - a prospective controlled study.

In contrast to multifollicular IVF, follicular flushing seems to increase the efficacy of monofollicular IVF treatments such as natural cycle IVF (NC-IVF). However, because follicular flushing causes loss of granulosa cells, it might negatively affect luteal phase length and endocrine function of the luteal body. A prospective cohort Phase II study was performed in 24 women undergoing NC-IVF. Women underwent a reference cycle with human chorionic gonadotrophin-induced ovulation without follicle aspiration and analysis of the length of the luteal phase and luteal concentrations of progesterone and oestradiol. In addition, they underwent a NC-IVF cycle which was performed identically but follicles were aspirated and flushed three times. The luteal phase was shorter in 29.2%, equal in 16.7% and longer in 50.0% of cases following flushing of the follicles. Overall, neither difference in luteal phase length was significant [median duration (interquartile range) in reference cycle: 13 (12; 14.5), IVF (flushing) cycle: 14 (12.5; 14.5), median difference (95% CI): 0.5 (-0.5 to 1.5)] nor median progesterone and oestradiol concentrations. In conclusion, follicular flushing in NC-IVF affects neither the length of the luteal phase nor the luteal phase concentrations of progesterone and oestradiol, questioning the need for luteal phase supplementation.

Preservation of Fertility in Patients with Cancer

PURPOSE: Advances in oncological treatment and in reproductive medicine have made family planning and fertility preservation after cytotoxic treatment more and more attractive. The attitude of patients towards fertility preservation is definitely positive. TECHNIQUES OF FERTILITY PRESERVATION: Basically, both medical treatments and invasive procedures are possible. Ovarian down-regulation by GnRH analogues is widely used and simple to perform, with little delay. However its efficacy has still not been definitely proven. Standard methods of ART require a stable partnership. Therefore, cryopreservation of unfertilized oocytes after hormonal stimulation might be better for most patients. In recent years, the introduction of new cryopreservation protocols allows acceptable results. Since hormonal stimulation is cycle dependent, it might cause a delay in oncologic treatment. New stimulation protocols, which can be initiated in every cycle phase are under investigation. Finally, ovarian biopsy and cryopreservation, followed by replantation might be an option. The first pregnancies and deliveries have been described. CONCLUSION: In most cases a multimodal approach must be offered to the patient. Most treatment modalities can be combined. To offer comprehensive treatment and allow the current techniques of fertility preservation to be evaluated, patients and doctors would benefit from networks such as like FertiProtekt, which guarantees nationwide patient support.

Die endometriale-trophoblastäre Interaktion: Einfluss von endometrialem Stroma auf die Genexpression des Cytotrophoblasten

Einleitung: Wahrend der Invasion des Trophoblasten in das mutterliche Endometrium erfolgen auf beiden Seiten multiple Veranderungen, welche in vivo nicht untersucht werden konnen. Mit Hilfe eines Co-Kulturmodells haben wir die Genexpression von Cytotrophoblastzellen nach Co-Kultur mit endometrialen Stromazellen und nach Kultur mit Uberstand von Schwangerschaftsdezidua analysiert, um den Einfluss des Endometriums auf Cytotrophoblast zu untersuchen. Material und Methoden: Explantate von Trophoblast des ersten Trimenons wurden in sechs verschiedenen Gruppen kultiviert. Zum Teil erfolgte die Kultur direkt auf Stromazellen zum anderen Teil mit Uberstand von Stromazellen sowie mit Uberstand von Dezidua. Nach 48h wurden die herausgewachsenen Cytotrophoblastzellen jeder Versuchsgruppe isoliert und die RNA extrahiert. Dann erfolgte die Real-Time PCR Untersuchung von Glycodelin, Hypoxia responsive factor–1, IGFBP–3, INSL–4, LOX und HSD11B2. Ergebnisse: In Cytotrophoblastzellen wird von den untersuchten Genen Glycodelin mRNA deutlich hochreguliert. Die hochste Regulation von Glycodelin wird bei der Ko-Kultur von Cytotrophoblast auf dezidualisierten endometrialen Stromazellen sowie bei der Kultur von Cytotrophoblast mit Uberstand von Schwangerschaftsdezidua beobachtet. Eine Regulation von anderen Genen im Cytotrophoblasten, wie Hypoxia responsive factor–1, IGFBP–1, INSL–4 LOX und HSD11B2, konnten wir nicht nachweisen. Schlussfolgerung: Ein direkter Einfluss von endometrialem Stroma und Dezidua auf den invasiven Cytotrophoblasten wird hier erstmalig anhand eines Gens gezeigt. Andere Cytotrophoblastgene mussen im Weiteren noch untersucht werden um ein genaueres Bild des Einflusses des Endometriums auf den Trophoblasten zu erhalten.

In-vitro-Maturation menschlicher Eizellen

Die erste Lebendgeburt nach einer In-vitro-Fertilisation (IVF) entstand nach Eizellentnahme in einem naturlichen Zyklus [38]. Die Verwendung von Gonadotropinen fuhrte zu der Effizienzsteigerung der IVF, die letztlich zu der Geburt von weltweit mehr als 1 Mio. Kindern gefuhrt hat. Allerdings birgt die Stimulation mit Gonadotropinen auch Risiken. So sind die Langzeiteffekte einer hoch dosierten Gonadotropinstimulation noch ungeklart und die Stimulation kann bei 1–5% der Patientinnen zu einer hochgradigen Uberstimulation mit erheblichen gesundheitlichen Risiken fuhren.