Peter Bielfeld

Regulation of embryonic implantation

The preimplantation embryo produces several factors during its development to signal its presence to the maternal organism. This paper will focus on the role of two distinctive cytokine and growth factor systems (interleukin-1 (IL-1) system and the vascular endothelial growth factor (VEGF) system) during early embryonic development and implantation. IL-1 receptor is expressed in the endometrium of various species and antagonising the biological effects of IL-1 leads to implantation failure in mice. We could show that this is due to an endometrial, not an embryonic effect. Furthermore, we could detect the expression of all components of the IL-1 system in preimplantation embryos from mice and humans. We could show a possible influence of IL-1 on other systems involved in embryonic implantation, including invasion (MMPs/TIMPs) and angiogenesis (VEGF), therefore suggesting a role of this cytokine family during early embryonic development. Immediately after contact to the endometrium, the embryo must induce angiogenesis to ensure its survival, VEGF is a potent angiogenetic growth factor. We have shown a cyclic regulation of the soluble VEGF-receptor, sflt, in human endometrium and have detected the expression of the transmembraneous VEGF-receptors, Flt-1 and kinase insert domain containing receptor (KDR) throughout the menstrual cycle. Furthermore, we have shown that the VEGF gene is one of the earliest genes activated during human preimplantation embryo development, giving rise to the assumption that VEGF is crucial for embryonic development.

Expression of vascular endothelial growth factor mRNA in human preimplantation embryos derived from tripronuclear zygotes

OBJECTIVE To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos. DESIGN Human preimplantation embryos not suitable for uterine transfer were examined for beta-actin and VEGF mRNA expression. Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion. SETTING Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Düsseldorf, Germany. PATIENT(S) Couples undergoing IVF by intracytoplasmic sperm injection for various reasons. INTERVENTION(S) Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion. MAIN OUTCOME MEASURE(S) Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA. RESULT(S) VEGF mRNA and protein could not be detected in unfertilized oocytes. However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16-cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts). The VEGF protein level was below the sensitivity of the ELISA. CONCLUSION(S) Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival.

Interferon gamma and interleukin 10 levels in preimplantation embryo culture media.

PurposeThe aim of our study is to elucidate whether human oocyteslembryos secrete IFNγand/or IL-10 and whether the fertilization process depends on the balance between these cytokines.MethodsA total of 142 embryo culture media from 24 patients were collected and the cytokine levels were tested with ELISA.ResultsIFNγand IL-10 were detectable in 40.1% and 29.6% of culture media respectively. The difference of IFNγand IL-10 levels in media from fertilized oocytes between day 1 and day 2 are significant (0.46 vs. 1.47 and 34.2 vs. 12.7, respectively). However there was no significant difference between the IFNγlevels of the media from fertilized and nonfertilized oocytes 0.46 vs. 0.85 at day 1 and 1.47 vs. 1.49 at day 2, as well as IL-10 levels 34.2 vs. 30.9 at day 1 and 12.7 vs. 9.58 at day 2 respectively.ConclusionsHuman preimplantation embryos secrete the cytokines IFNγand IL-10. No effect of these cytokines on fertilization process could be shown.

Interferon‐Gamma Production by the Human Preimplantation Embryo

PROBLEM: This study demonstrated that the human embryo produces interferon‐gamma (IFNγ). It is important to know whether IFNγ can be produced before implantation. Therefore the aim of this study was to evaluate the profile of IFNγ production between days 2 and 5 after in vitro fertilization.

Einfluß der assistierten Reproduktion auf die Inzidenz von Mehrlingsschwangerschaften

Zum ThemaDurch die Hormonstimulation der Ovarien im Rahmen der Sterilitätstherapie und der assistierten Reproduktionmedizin ist die Inzidenz von Mehrlingsschwangerschaften um das 20 fache gegenüber der natürlichen Inzidenz gestiegen. Obwohl 25 % aller Schwangerschaften nach Maßnahmen der assistierten Reproduktion in Deutschland Mehrlingsgraviditäten sind, entsteht der größere Anteil im Rahmen von ovariellen Stimulationstherapien mit In-vivo-Konzeptionen. Da in all diesen Schwangerschaften eine iatrogene Beteiligung ursächlich gegeben ist, sollten diese Therapieformen nur von speziell ausgebildeten Ärzten verantwortlich durchgeführt werden. Neben Faktoren wie Alter, Parität und Sterilitätsanamnese der Gravida stellt die Mehrlingsgravidität den entscheidenden Risikofaktor für mütterliche und fetale bzw. neonatale Komplikationen dar. Zusätzliche negative Einflüsse sind durch eine Zunahme an monochorialen Geminigraviditäten nach Sterilitätstherapien zu erwarten. Aus diesen Gründen ist der Prävention der Entstehung von Drillingsgraviditäten und vor allem höhergradigen Mehrlingsschwangerschaften, bei allem therapeutischen Bemühen um die Erfüllung des Kinderwunsches, Priorität einzuräumen.

Fertilizing capacity of various populations of spermatozoa within an ejaculate

To determine whether ejaculates consist of various populations of sperm with varying degrees of fertilizing capacity, semen was filtered through a glass-wool column and the filtrate aliquoted sequentially into three portions. The ejaculate and its respective filtrates were evaluated for progressive motility, ability to respond to hypoosmotic swelling (HOS) test, acrosin content, ability to capacitate and acrosome react (AR), and ability to penetrate into denuded hamster oocytes (SPA). Filtrate 1 contained significantly more sperm with progressive motility, increased acrosin content, and positive HOS and SPA results than did the ejaculate. AR did not differ between the ejaculates and the respective filtrates. The data suggest that the ejaculate consists of various populations of sperm with different properties and that these populations may be separated by glass-wool filtration.

Influence of media and thermal shock on sperm capacitation

Summary. To elucidate the influence of medium and thermal shock in inducing sperm capacitation, eight ejaculates were processed as follows. Each ejaculate was divided into three aliquots. To two of the aliquots, an equal volume of saline was added, while TEST‐yolk was added to the remaining aliquot. All three samples were cooled slowly to 5 °C and incubated for 18–20 h. After aspirating the supernatant, 6 ml of Tyrode solution containing 0.06 g% egg yolk, warmed to 37 °C was added without disturbing the sperm pellet to the aliquot containing TEST‐yolk and to one of the aliquots mixed with saline. To the remaining saline‐processed sperm pellet, 6 ml TEST‐yolk (37 °C) was added. All samples were washed twice (400 × g for 5 min) and processed for the sperm penetration assay (SPA). Samples, either pre‐incubated in TEST‐yolk (mean ± SEM; 65.9 ± 11.7%) or warmed (thermal shock) with TEST‐yolk (62.6 ± 12.1%) yielded significantly (P<0.05) higher SPA outcome than the samples processed and warmed with saline (17.8 ± 12.0%). The penetration indices were not statistically different from each other. It therefore appears that the medium influences the sperm capacitation process to a greater extent and may augment the influence of thermal shock.

A new CentriSwim procedure to increase the recovery of motile spermatozoa.

Summary. A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim‐up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim‐up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory‐induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim‐up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2‐cm radius upward‐directing arm, at an angle of 60° for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22–24°C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory‐induced oligoasthenozoospermic specimens were significantly higher than following standard swim‐up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim‐up technique.

Einfluß von parakrinen Faktoren auf die embryonale Implantation

Zum ThemaDer Eintritt einer intrauterinen Schwangerschaft beim Menschen nach erfolgreicher Befruchtung beruht auf 2 grundlegenden Voraussetzungen: der physiologischen Reifung des Embryos während der 4–5 Tage des Transports durch den Eileiter und der zeitgerechten Entwicklung eines rezeptiven Endometriums. Der Präimplantationsembryo bildet verschiedene Proteine, um seine Präsenz dem mütterlichen Körper anzuzeigen und die regelrechte Interaktion zwischen Embryo und Endometrium wird – zumindest teilweise – durch parakrine Zytokine und Wachstumsfaktoren gesteuert. Die Erforschung dieser Zusammenhänge könnte zur Verbesserung der In-vitro-Kulturbedingungen menschlicher Embryonen führen und u. U. die Implantations- und Schwangerschaftsraten im Rahmen der assistierten Reproduktion erhöhen. Die folgenden Ausführungen sollen einerseits einen Überblick über die aktuelle Literatur geben und auf der anderen Seite einige neue, experimentelle Modelle beschreiben sowie deren zukünftige Perspektiven für die assistierte Reproduktion erläutern.

No change in acrosome reaction of human spermatozoa during storage in cervical mucus

Summary. Incubation of human spermatozoa in capacitation media for 24 h results in a significant increase in acrosome reacted spermatozoa as compared to preincubation (baseline) levels. By contrast, sperm samples recovered from the cervix for as long as 72 h after coitus only showed baseline percentage of acrosome reacted spermatozoa. These results indicate that spermatozoa do not undergo the acrosome reaction during cervical storage and suggest that cervical mucus suppresses the spontaneous acrosome reaction of spermatozoa that become capacitated in the cervix.