Objective To compare the assessment of endometrial maturation parameters in endometrial secretion samples obtained by a novel minimally invasive technique with those assessed in tissue biopsies.Design Prospective study.Setting University Hospital.Population Healthy female volunteers attending a gynaecological outpatient clinic.Methods Endometrial secretion fluid and tissue sampling 5 days after a spontaneous ovulation assessed with ultrasound.Main outcome measures Progesterone (P) receptor, Ki‐67 expression and the Noyes criteria were used to date endometrial biopsies. In the endometrial fluid samples, glycodelin A (GdA), leukaemia inhibitory factor (LIF) and P levels were analysed, and protein content and electrophoresis patterns were determined.Results All data were correlated to estradiol (E2) and P serum concentrations. The dating according to histology and immunohistochemical staining patterns correlated significantly with GdA levels (r = 0.376, P =0.048) in endometrial fluid samples as well with serum levels of E2 (r = 0.568, P =0.001) and P (r = 0.408, P =0.023). No correlation was observed between tissue dating and LIF levels and protein content in endometrial fluid samples.Conclusions The measurement of GdA in endometrial secretion samples may provide a less invasive method for assessing endometrial maturation in potential conception cycles without disrupting implantation.

The objective of this study was to investigate the effect of ovarian stimulation for IVF on endometrial secretion and tissue markers of receptivity in the mid-luteal phase. In 10 oocyte donors, endometrial secretions and biopsies were sampled 5 days after spontaneous ovulation and oocyte retrieval in consecutive cycles. Four subjects received progesterone in the luteal phase of the stimulated cycles. Mid-luteal endometrial maturation in the stimulated cycle was compared with the spontaneous cycle, by histological dating, Ki-67, oestrogen receptor (ER) and progesterone receptor (PR) expression, secretion levels of leukaemia inhibitory factor (LIF), glycodelin A (GdA) and progesterone, and protein profile. No significant differences in histological markers, expression of Ki-67, PR, ER, secretion protein profiles or concentrations of LIF, GdA, or progesterone were observed when comparing natural with stimulated cycles. Progesterone supplementation of stimulated cycles was associated with significantly lower Ki-67 (P = 0.03) and ER (P = 0.04) expression compared with the non-supplemented stimulated cycle. In this pilot study, ovarian stimulation was not demonstrated to alter the studied markers of endometrial maturation in the mid-luteal phase.

Analysis of protein patterns in endometrial secretion fluid may offer a relatively non-invasive means of assessing endometrial receptivity during fertility treatment cycles. In order to study the impact of the removal of endometrial secretions on embryo implantation, a prospective matched controlled study was performed. In 66 women undergoing IVF, endometrial fluid was obtained transcervically by aspiration just prior to embryo transfer (study group). Biochemical and ongoing pregnancy rates were compared with 66 control patients matched for stimulation treatment protocol, age, number of collected oocytes and number of high quality embryos. The protein content and uterine fluid protein profile in each sample was determined. Respective biochemical and ongoing pregnancy rates per embryo transfer were 36 and 33% in patients who underwent aspiration of endometrial secretion, compared with 33 and 30% respectively in matched control patients (P = 0.84 and P = 0.85). The protein content in endometrial fluid was sufficient for protein pattern analysis. Uterine fluid aspiration prior to IVF embryo transfer is a safe method for obtaining sufficient material for uterine secretion electrophoresis, thus allowing analysis of protein patterns serving as receptivity markers during treatment cycles. This technique may offer a novel tool for assessing endometrial receptivity during treatment cycles without affecting implantation rates.

After its original description as a steroid-dependent protein in the rabbit uterus, uteroglobin became one of the best characterized proteins. However, detailed knowledge of its physiological role remains an enigma. in this study we investigate how its structure is phylogenetically conserved in the horse compared to other mammalian species. Northern blot analysis showed that in horses, the main expression of uteroglobin appears in lung, uterus, and prostate tissues. Western blot analysis demonstrated that the dimeric form of uteroglobin is found predominantly in biological compartments. Using a RACE-PCR technique, we cloned and sequenced the full-length cDNA (473 base pairs) that encodes equine uteroglobin. The nucleotide sequence was shown to characterize the primary structure of this protein. This enabled us to add equine uteroglobin to a comparative amino acid alignment of 8 other uteroglobin molecules, and finally, to unravel 14 evolutionary completely conserved amino acids. We summarize these results with a computer-based 3-D model of horse uteroglobin, and discuss new concepts on the physiological role of uteroglobin, in particular as a specific binding protein.

The contemporary approach to ovarian stimulation for IVF treatment results in supraphysiological concentrations of steroids during the follicular and luteal phases of the menstrual cycle. These sex steroids act directly and indirectly to mature the endometrium, influencing receptivity for implantation. Corpus luteum function is distinctly abnormal in IVF cycles, and therefore luteal support is widely used. Various reasons may underlie the defective luteal phase, including (i) ovarian hyperstimulation per se, (ii) gonadotrophin-releasing hormone (GnRH) analogue co-treatment and (iii) the use of human chorionic gonadotrophin (HCG) to induce final oocyte maturation. The recent introduction of GnRH antagonist co-treatment for the prevention of a premature LH rise during the late follicular phase allows for different approaches to ovarian stimulation for IVF. However, a recent meta-analysis showed that implantation rates may be compromised by using GnRH antagonists in currently employed regimens. The development of endometrium receptive to embryo implantation is a complex process and may be altered by inappropriate exposure to sex steroids in terms of timing, duration and magnitude. New approaches to the assessment of endometrial receptivity are now required. Novel approaches to ovarian stimulation aimed at adjusted GnRH antagonist regimens and achieving a more physiological luteal phase endocrinology are now appearing in the literature and may represent an important step in the improvement of the overall health economics of IVF.

The biological aim of the differentiation and maturation of endometrial tissue compartments during any menstrual cycle is the achievement of suitable conditions for blastocyst implantation and the establishment of pregnancy. Infertility and early embryonic loss are frequently caused by insufficient endometrial differentiation. Even any incomplete receptivity stage of the luteal phase endometrium will prevent attachment and implantation. We have studied the physiological changes throughout an endometrial cycle to elucidate causes of endometrial insufficiency leading to subfertility or infertility. Up to now, the histological changes described by Noyes et al. are understood as classical diagnostic approaches. However, evidence is accumulating that molecular deficits of endometrial differentiation are by no means detectable histologically, and consequently ask for the research on new diagnostic methods and parameters. There are histochemical localizations of specific protein molecules, adhesion molecules and cytokines, which permit by far more detailed and significant molecular analyses than any classical morphological means could yield. Moreover, there are convincing arguments to use further biochemical assessments on proteins of the uterine secretions as specific diagnostic parameters. The electrophoretical resolution presents typical protein patterns, which in turn can be interpreted as characteristic reflexions of the functional phases of the endometrial cycle. What is demonstrated as the so-called adequate luteal phase protein pattern clearly is the product of the receptive endometrium, reflecting the "implantation window". This is established already two days after ovulation and persists usually eight further days, if the endometrial cycle is undisturbed (15th to 24th day of the cycle).

Zusammenfassung Biologisch ist die Reifung des Endometriums in jedem Zyklus darauf gerichtet, einer Blastozyste günstige Bedingungen für die Implantation zu bieten. Infertilität und frühes Ende einer Schwangerschaft beruhen nicht selten auf einer unvollkommenen Reifung des Endometriums. Bereits eine mangelhafte Rezeptivität des Endometriums führt zum Scheitern der Implantation. Um die endometriale Ursache einer Infertilität zu erkennen, werden die normalen zyklischen Veränderungen des Endometriums studiert. Morphologische Veränderungen bilden bis heute in diesem Bemühen der Gynäkologen noch die Grundlage der klassischen histologischen Diagnostik nach Noyes et al. (1950). Da jedoch evident ist, dass molekulare Defizite des Endometriums sich nicht unmittelbar histologisch erfassen lassen, werden neue Methoden und signifikante Parameter gesucht, die diese Art funktioneller Defekte diagnostisch erfassen lassen. Histochemische Lokalisierungen von definierten Proteinen, von Adhäsionsmolekülen und Zytokinen, erlauben bereits eine detailliertere und aussagekräftigere molekulare Analyse als es die klassische Morphologie ermöglicht. Überzeugende Argumente sprechen inzwischen dafür, mit einfachen biochemischen Analysen die Proteine des Uterussekrets als weitergehende signifikante Parameter zu nutzen. Die elektrophoretische Auftrennung des Uterussekrets ergibt typische Proteinmuster, welche die zyklischen Funktionsphasen des Endometriums darstellen. Das adäquate Proteinmuster der Lutealphase ist als das Produkt eines rezeptiven Endometriums zu betrachten und repräsentiert das für diese molekulare Analytik charakteristische „Implantationsfenster“. Es wird bereits 2 Tage post ovulationem exprimiert und bei ungestörtem Zyklusverlauf 8 Tage aufrechterhalten (15.–24. Zyklustag).

Leptin and its receptor are involved in endocrine and paracrine regulation of metabolism, obesity and reproduction. Here, we describe the detection of the functional long isoform receptor of leptin in human endometrium. The leptin receptor protein was shown to be expressed in glandular and luminal epithelium and is periodically regulated throughout the menstrual cycle, demonstrating main expression in follicular and mid-luteal phase. In contrast, leptin receptor mRNA is detectable by reverse transcription-polymerase chain reaction (RT-PCR) as a constitutive component. Since RT-PCR analyses showed that leptin is not expressed in this tissue, the present study suggests that the human endometrium is a novel target for leptin. Therefore, we investigated 11 subfertile patients who underwent two biopsies in one menstrual cycle. The patients presented with a repetitive endometrial maturation defect, but showed adequate serum hormone concentrations and normal steroid hormone receptor expression and down-regulation in the endometrium. These patients were, however, deficient for expression of the functional leptin receptor. These analyses provide evidence that the lack of the leptin receptor in an ovulatory cycle may contribute to subfertility by a yet undefined 'endometrial factor'.

Annals of the New York Academy of SciencesVolume 923, Issue 1 p. 332-335 Uteroglobin Expression and Release in the Human Endometrium F. MÜLLER-SCHÖTTLE, Corresponding Author F. MÜLLER-SCHÖTTLE Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, Germanya Address for correspondence: Department of Anatomy and Reproductive Biology, RWTH University of Aachen, Wendlingweg 2, 52057 Aachen, Germany. Voice: +49-241-8089 110; fax: +49-241-8888 508. fmueller-schoettle@post.klinikum.rwth-aachen.deSearch for more papers by this authorI. CLASSEN-LINKE, I. CLASSEN-LINKE Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, GermanySearch for more papers by this authorK. BEIER-HELLWIG, K. BEIER-HELLWIG Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, GermanySearch for more papers by this authorK. STERZIK, K. STERZIK Institute for Reproductive Medicine, Ulm, GermanySearch for more papers by this authorH. M. BEIER, H. M. BEIER Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, GermanySearch for more papers by this author F. MÜLLER-SCHÖTTLE, Corresponding Author F. MÜLLER-SCHÖTTLE Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, Germanya Address for correspondence: Department of Anatomy and Reproductive Biology, RWTH University of Aachen, Wendlingweg 2, 52057 Aachen, Germany. Voice: +49-241-8089 110; fax: +49-241-8888 508. fmueller-schoettle@post.klinikum.rwth-aachen.deSearch for more papers by this authorI. CLASSEN-LINKE, I. CLASSEN-LINKE Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, GermanySearch for more papers by this authorK. BEIER-HELLWIG, K. BEIER-HELLWIG Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, GermanySearch for more papers by this authorK. STERZIK, K. STERZIK Institute for Reproductive Medicine, Ulm, GermanySearch for more papers by this authorH. M. BEIER, H. M. BEIER Department of Anatomy and Reproductive Biology, RWTH University of Aachen, 52057 Aachen, GermanySearch for more papers by this author First published: 25 January 2006 https://doi.org/10.1111/j.1749-6632.2000.tb05544.xCitations: 9Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume923, Issue1THE UTEROGLOBIN/CLARA CELL PROTEIN FAMILYDecember 2000Pages 332-335 RelatedInformation

Tumour necrosis factor-alpha (TNF-alpha is a pleiotropic cytokine synthesized throughout the female reproductive tract. Even though evidence has accumulated that supports its role in autocrine and paracrine processes, its expression and function in the human endometrium are still not completely understood. To gain a better understanding of the synthesis and release of TNF-alpha in the endometrium and how this relates to concentrations in uterine secretion, its expression throughout the menstrual cycle was investigated by three different techniques. Samples of endometrial tissue and uterine secretions were collected from patients undergoing abdominal and vaginal hysterectomy for benign reasons. The mRNA expression of TNF-alpha was investigated in homogenized endometrial tissue by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) (n = 18). An assessment of the cellular TNF-alpha protein localization in the endometrial glands was performed immunohistochemically (n = 39). The concentrations of the secreted TNF-alpha protein in endometrial secretion were determined by enzyme-linked immunosorbent analysis (n = 30). All three methods gave similar results on the temporal expression of TNF-alpha mRNA and TNF-alpha protein during the cycle. Concentrations of endometrial TNF-alpha mRNA in tissue samples and TNF-alpha protein in uterine secretion were quite low at the beginning of the cycle, rose sharply in the mid- to late proliferative phase and decreased towards the end of the cycle. The concentrations of TNF-alpha protein in the endometrial glands, as shown by immunohistochemical investigation, stayed high throughout the secretory phase at values slightly below those of the late proliferative phase.

Uteroglobin is a progesterone binding protein, a member of the antiflammin gene family and possibly a novel cytokine. Initially, uteroglobin was identified as the major protein of rabbit uterine secretion during the phase of preimplantation. Counterparts of the rabbit uteroglobin or its gene are described in rat, mouse, hamster, hare, pig, horse and human. While uteroglobin appears as one of the most extensively studied proteins, particularly its physico-chemical properties, including its crystal structure and its gene, the true physiological role of this protein still remains to be unravelled. Essential to understanding the significance of human uteroglobin in reproductive organs, particularly in the endometrium, is a knowledge of the spatial and chronological expression of this secretory protein. Our studies on 115 volunteers combined reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and quantitative assessment by an enzyme-linked immunosorbent assay for uteroglobin. The expression, localization and release of uteroglobulin in the human endometrium are presented. Secretory uteroglobin is found in endometrial tissue homogenates in highest levels of expression during the early luteal phase (days 15-19, 340 pg/mg total protein). In turn, uteroglobin is released into the uterine lumen in peak amounts during the receptive phase of the menstrual cycle (mid-luteal phase, days 20-23, secretion level 833.4 pg/mg total protein). Our immunohistochemical studies match with these results, as uteroglobin is located during the early and mid-luteal phase in the apical compartments of endometrial gland cells. These observations strongly suggest an involvement of uteroglobin in endometrial preparations for implantation.

The protein composition of human uterine secretions was studied by SDS polyacrylamide gradient gel electrophoresis (SDS-PAGE). Material and methods: 278 secretion samples from 246 hormonally stimulated or unstimulated patients treated for infertility or cervical dysplasia were collected at different phases of the menstrual cycle by sampling transcervically uterine secretions. The proteins of the samples were studied by SDS-PAGE and by densitometric analysis of the electrophoretic protein patterns. Expression of certain proteins were evaluated, mathematically analysed and semiquantitatively described as a reproducible and comparable parameter. Results: The analysis reveals changing protein patterns during the menstrual cycle: An increasing number of small protein bands between 15 kDa and 60 kDa appears during the proliferative and the early and midsecretory phases. In the secretory phase the protein pattern is completed by three characteristic bands between 12.5 kDa and 21.0 kDa, known as histones HZ A, H2 B and H3. This results in a maximal expression of the protein pattern during the mid-secretory phase, the time of implantation. Analysis of the protein patterns in relation to infertility reveals weaker protein expression in patients suffering from tubal occlusion and idiopathic infertility in comparison to those apparently fertile. Conclusion: This study supports the results of an earlier study by Beier-Hellwig et al. (1989) where characteristic changes of the protein patterns during the menstrual cycle were evaluated by a visual and subjective analysis. The introduction of a semi-quantitative analysis of the protein patterns leads to a reproducible description of the fine tuning of the protein patterns during the cycle. This technique promises to be useful as a diagnostic tool for the analysis of endometrial function.

Fragestellung: Es sollte geprüft werden, ob in Abhängigkeit von der Zyklusphase, der Östradiol- und Progesteronserumspiegel sowie der Infertilitätsursache elektrophoretische Bandenmuster mittels reproduzierbarer Parameter beschrieben werden können, die einen Vergleich verschiedener Sekretauftrennungen erlauben. Material und Methodik: Von 246 Patientinnen aus der Sterilitäts- und Zervixdysplasiesprechstunde wurden 278 Uterussekretproben transzervikal abgenommen, mittels der SDS-Polyacrylamid-Gradienten-Gel-Elektrophorese (SDS-PAGE) aufgetrennt und die resultierenden Proteinbandenmuster analysiert. Neben der Darstellung und Analyse einzelner markanter Proteinbanden wird die Expression eines ganzen Bandenspektrums nach der densitometrischen Vermessung mathematisch beschrieben und semi-quantitativ als Zahlenwert dargestellt. Ergebnisse: Im Verlauf des Zyklus kann eine graduelle Veränderung der Bandenmuster herausgearbeitet werden: Während der Proliferationsphase und der frühen und mittleren Sekretionsphase wird eine zunehmende Anzahl kleiner Proteinbanden zwischen 15 kDa und 60 kDa exprimiert. In der Sekretionsphase wird das Bandenmuster durch die Expression eines markanten Bandentripletts, bestehend aus den Histonproteinen H2A, H2B und H3 zwischen 12,5 kDa und 21,0 kDa ergänzt. Das Uterussekretbandenmuster ist in Abhängigkeit vom Progesteronserumspiegel in der mittleren Sekretionsphase, dem Zeitpunkt der Implantation, maximal ausgeprägt. Beschrieben werden des weiteren Veränderungen der Bandenmuster in Abhängigkeit von der Infertilitätsursache. Patientinnen mit einem Tubenfaktor oder einer idiopathischen Infertilitätsursache weisen ein schwächer entwickeltes Bandenmuster auf als jene, die als fertil anzusehen sind. Schlußfolgerung: Die Untersuchungen dieser Arbeit untermauern und ergänzen die Erkenntnisse aus den Arbeiten von Beier-Hellwig et al. (1989): Durch eine visuelle und subjektive Beurteilung der Sekretbandenmuster wurden charakteristische Veränderungen der Musterausprägung während des Menstruationszyklus dargestellt und mit endometrialen Funktionszuständen korreliert. Durch die in dieser Arbeit dargestellte Semi-Quantifizierung der Proteinbanden konnte die Modulation der Musterausprägung im Verlauf des Zyklusgeschehens und in Abhängigkeit von verschiedenen Einflußgrößen mittels reproduzierbarer Parameter mathematisch nachvollzogen werden. Dieses rechnerische Vorgehen erlaubt die Analyse des Sekretbandenmusters und die Anwendung der Sekretanalyse bei der Funktionsdiagnostik des Endometriums.

Endocrine and paracrine controls regulate the endometrium during the luteal phase of the cycle to permit implantation. Part of this differentiation process is the production of a specific secretion which fills the intrauterine cavity and glandular lumen. Its molecular composition originates from the gland secretion, from transudations from stroma, from the endometrial blood vessels, and last, but not least, from cellular components of apoptotic and exfoliated cells. We have studied the secretions of all phases during the menstrual cycle using patterns evaluated by SDS-PAGE, by laser densitometry or Western blots. Uterine secretion electrophoresis (USE) permits detailed analyses of the intrauterine micromilieu and allows clinical assessment of the receptive stage of endometrium during the luteal phase. Several individual protein bands have been defined as characteristic markers for such receptive pattern. We have isolated and identified the molecular structure of several of these proteins, e.g. histones, cyclophilin, transthyretin, haptoglobin and uteroglobin. Investigations on the endocrine regulation of these proteins, were carried out on the uterine secretions of patients treated with progesterone antagonists (mifepristone and onapristone). The results demonstrate how progesterone-dependent components produce a receptive pattern, which can serve as a useful and precise marker in the clinical diagnosis of the luteal phase. Essential progesterone-dependent components differentiating during the luteal phase may provide new targets for contraceptive interventions by preventing the physiological changes typical of receptivity.

The protein composition of human uterine secretions was studied by high-resolution two-dimensional (2-D) electrophoresis. Secretions were collected at different phases of the menstrual cycle by sampling directly intraluminal material from 25 patients undergoing abdominal or vaginal hysterectomies. The proteins of the samples and the patients' serum were separated by isoelectric focusing, using immobilised pH gradients (IPGs, pH 4-8), followed by horizontal SDS polyacrylamide gradient gel electrophoresis (kDa 5 - 150) and silver staining. Out of a total of more than 500 spots we defined 23 spots in the protein patterns which could not be found in the patients' serum. Four spots seemed to fluctuate in relation to the cycle: the first spot (pI 5.5, kDa 31) was maximally expressed in the secretory phase, the second one (pI 7.0, kDa 31) perimenstrually in the early proliferative and late secretory phase, the third one (pi 6.4, kDa 22) in the secretory and the fourth one (pI 4.7, kDa 11) in the late proliferative to midsecretory phases. The second spot was N-terminally sequenced following micropreparative 2-D electrophoresis with narrow range IPGs. The protein could be identified as a fragment of serum albumin which was produced by cleavage between the hydrophobic amino acid leucine and arginine. It is possible that the matrix metalloproteinase MMP-7 caused the fragmentation. MMP-7 preferentially cleaves bonds of hydrophobic character, it is expressed in the endometrial glands and it can be detected in high concentrations at the beginning and the end of the menstrual cycle. Since the protein map has been obtained with a highly reproducible 2-D gel system, it will be comparable to studies from other laboratories with a high degree of reliability.