Christian De Geyter

Prospective evaluation of the ultrasound appearance of the endometrium in a cohort of 1,186 infertile women

OBJECTIVE To investigate the effect of differences in endometrial thickness and pattern as visualized with present-day high-resolution transvaginal ultrasound equipment on the outcome of assisted reproductive treatment. DESIGN Prospective cohort study. SETTING Two large infertility units in university hospitals. PATIENT(S) The endometrial characteristics of 981 patients during 1,600 assisted reproductive treatment cycles were compared with those of 205 untreated women. In addition, abnormal echogenic patterns of the endometrium were characterized histologically in 44 patients. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Pregnancy rates. RESULT(S) The endometrium was significantly thinner in untreated women and women treated with IUI than in women treated with IVF or intracytoplasmic sperm injection. The exponential proliferation of the endometrium was similar in conception and nonconception cycles. The odds for a successful pregnancy were significantly lower in the presence of a thin endometrium in women treated with IUI but not in women treated with the long stimulation protocol for IVF or intracytoplasmic sperm injection. Singleton pregnancies were more common than multiple pregnancies in patients with thin endometria. CONCLUSION(S) The pregnancy rates of assisted reproductive procedures are influenced only marginally by the degree of endometrial proliferation, and treatment should not be canceled because of inadequate endometrial thickness.

The Multipotency of Luteinizing Granulosa Cells Collected from Mature Ovarian Follicles

Graafian ovarian follicles consist of follicular fluid, one single mature oocyte, and several hundred thousands of granulosa cells (GCs). Until now, luteinizing GCs have been considered to be terminally differentiated, destined to undergo death after ovulation. Present concepts of luteal function, endocrine regulation of early pregnancy, and the recruitment of new ovarian follicles are all based on the cyclical renewal of the entire population of GCs. We now demonstrate that luteinizing GCs isolated from the ovarian follicles of infertile patients and sorted with flow cytometry based upon the presence of their specific marker, the follicle‐stimulating hormone receptor (FSHR), can be maintained in culture over prolonged periods of time in the presence of the leukemia‐inhibiting factor (LIF). Under those conditions the markers of GC function such as FSHR and aromatase gradually disappeared. POU5F1 (POU domain, class 5, homeobox 1), a typical stem cell marker, was expressed throughout the culture, but germ line cell markers such as nanog, vasa, and stellar were not. Mesenchymal lineage markers such as CD29, CD44, CD90, CD105, CD117, and CD166, but not CD73, were expressed by substantial subpopulations of GCs. The multipotency of a subset of GCs was established by in vitro differentiation into other cell types, otherwise not present within ovarian follicles, such as neurons, chondrocytes, and osteoblasts. Follicle‐derived stem cells were also able to survive when transplanted into the backs of immunoincompetent mice, in vivo generating tissues of mesenchymal origin. The unexpected findings of multipotency of cells with prolonged lifespans originating from ovarian follicles are likely to have a significant impact on evolving theories in ovarian pathophysiology, particularly with reference to ovarian endometriosis and ovarian cancer. STEM CELLS 2009;27:210–219

Retinol-Binding Protein 4 in Polycystic Ovary Syndrome—Association with Steroid Hormones and Response to Pioglitazone Treatment

CONTEXT Polycystic ovary syndrome (PCOS) is frequently associated with insulin resistance. OBJECTIVE The aim of the study was to investigate a putative role of the adipokines retinol-binding protein 4 (RBP4), adiponectin, and visfatin in a cohort of patients with PCOS and their response to treatment with pioglitazone. DESIGN AND SETTING We conducted a randomized, controlled, double-blind study at a tertiary referral center. PATIENTS AND INTERVENTIONS Forty premenopausal women with PCOS were allocated to receive treatment with either pioglitazone (30 mg/d) or a placebo for a period of 3 months. MAIN OUTCOME MEASURES Serum concentrations of RBP4, adiponectin, and visfatin were determined along with metabolic and hormonal parameters before and after treatment. RESULTS Serum adiponectin concentrations were higher after treatment with pioglitazone (P = 0.003), whereas RBP4 levels tended to decrease (P = 0.06), and visfatin concentrations remained unchanged. We found RBP4 serum concentrations at baseline to be positively correlated with serum levels of testosterone (R = 0.446; P = 0.005), 17-OH progesterone (R = 0.345, P = 0.037), and dehydroepiandrosterone sulfate (R = 0.347; P = 0.041). However, these correlations were abolished after treatment with pioglitazone. Patients with high RBP4 levels had significantly higher hirsutism scores (P = 0.038 before and P = 0.034 after treatment). In contrast, serum adiponectin concentrations were related to parameters of impaired glucose metabolism, and no significant associations were detected for visfatin. CONCLUSIONS Our results suggest that RBP4 may contribute to endocrine changes and to the phenotypic manifestation of patients with PCOS because higher RBP4 concentrations are associated with higher androgen levels and higher clinical hirsutism scores independently of pioglitazone treatment. The molecular involvement of RBP4 in human steroid metabolism requires further clarification.

Differences in low-grade chronic inflammation and insulin resistance in women with previous gestational diabetes mellitus and women with polycystic ovary syndrome

Background. Polycystic ovary syndrome (PCOS) and gestational diabetes mellitus (GDM) are both characterized by an increase in insulin resistance. Our goal in the present study was to measure insulin resistance (as estimated by homeostasis model assessment, sex hormone-binding globulin (SHBG) and adiponectin concentrations) and parameters of low-grade inflammation in non-diabetic, non-hyperandrogenic ovulatory women with previous GDM (pGDM) and in non-diabetic women with classic PCOS, characterized by hyperandrogenism and oligo/anovulation. Patients and design. We evaluated 20 women with PCOS, 18 women with pGDM and 19 controls, all matched according to body mass index (BMI). Fasting blood samples were drawn in all women 3–6 days after spontaneous or dydrogesterone-induced withdrawal bleeding. Body fat distribution was assessed using dual-energy X-ray absorptiometry in all women. Results. After adjusting for age and percent body fat, measures of insulin resistance such as SHBG and adiponectin concentrations were decreased and central obesity was increased in women with PCOS and pGDM compared with controls (all p < 0.05). Parameters of low-grade inflammation such as serum tumor necrosis factor-α and highly sensitive C-reactive protein concentrations, white blood cell and neutrophil count were increased only in women with PCOS compared with BMI-matched controls (all p < 0.05). Conclusions. Certain markers of insulin resistance are increased in both women with PCOS and women with pGDM, while low-grade inflammation is increased only in PCOS. PCOS and GDM might represent specific phenotypes of one disease entity with an increased risk of cardiovascular disease, whereby women with PCOS demonstrate an augmented cardiovascular risk profile.

Evolutionarily conserved Bok proteins in the Bcl-2 family

Apoptosis is under tight control of rather complex regulatory circuits in which proteins of the Bcl-2 family act as a critical life^death decision point. The Bcl-2 family consists of a growing number of proteins [1]. In Caenorhabditis elegans there is only one member, ced-9, existing. In mammalian cells the regulation of apoptotic process by the Bcl-2 family appears to be very complex. More than 10 members of the Bcl-2-related proteins have been identi¢ed so far. Previously, we cloned a human Bok gene (Hbok) from a human infant brain library (GenBank accession number AF174487). By search of the expressed sequence tags (EST) database/Drosophila genome sequence database using the amino acid sequences of HBcl-xL and HBok, and by use of polymerase chain reaction (PCR), we further identi¢ed two Drosophila and a chicken Bok homologue (Fig. 1). Brie£y, a Blast Search of the GenBank database ESTs was performed and a partial nucleotide sequence of a Drosophila cDNA was identi¢ed. Subsequently, 5P-RACE PCR was performed on mRNA of Drosophila Schneider L2 (S2) cells to amplify the 5P sequences, which were then to be compared to genomic sequences from Flybase. This resulted in obtaining the entire coding region containing a 978 nt open reading frame (Fig. 1A). The predicted amino acid sequence showed 35% identity to the human Bok protein, but about 25% identity to other Bcl-2-related proteins. This gene was then named Dbok. Dbok is identical to Drob-1 recently published by Igaki et al. [2], but longer than Debcl from Colussi et al. [3]. Alignment of cDNA sequence along with genomic sequences from the Drosophila genome sequence database revealed that the genomic sequence of Dbok contains three exons. This result and a further search of the Drosophila genomic sequence database recovered a sequence which represents another distant Bcl-2-related gene, named Dbx (Fig. 1A). In addition, we identi¢ed a chicken Bok from three chicken cDNA clones, which contain partial nucleotide sequences. Sequence analysis resulted in identi¢cation of a 1158 nt fulllength cDNA which contains a 641 nt coding sequence. Its amino acid sequence shares 81% identity with that of human Bok (Fig. 1A). The analysis of Bok proteins with other members in the Bcl-2 family revealed that they contain all four di¡erent BH domains, BH1, BH2, BH3 and BH4 (Fig. 1A). But both Drosophila homologues contain two BH4 domains, whereas chicken and human Bok contain only one BH4 domain (Fig. 1B). The alignment shows that a highly conserved stretch of amino acids, ITWGK, is present in the BH1 domain. It di¡ers in this region from other Bcl-2 family members and is a characteristic for this sub-group in the Bcl-2 family (Fig. 1A). Dbok has a long N-terminus with an additional BH4 domain as compared to other Bcl-2 related proteins. The topology prediction of the membrane regions of Dbok showed that addition to a candidate membrane-spanning segment of amino acids 221^241 (BH1 domain) and a putative hydrophobic membrane anchor between amino acid 290 and 310, both of which have been shown to have important features, the Nterminal sequence also contains a transmembrane segment within amino acids 14^34 [4]. This region is not present in vertebrates. To explore the structure and function relationship of Dbok, we have used an apoptosis assay, in which apoptotic REF52 cells round up at an early apoptotic stage and detach from the surface of the dish [5]. The DNA encoding fulllength Dbok was cloned into the pcDNA3 expression vector. The constructs were transfected into REF52 rat ¢broblasts together with a vector (pEGFP-N1) encoding the green £uorescent protein (GFP). GFP served as marker for successfully transfected cells. 15 h after transfection, apoptotic REF52 cells were counted under a £uorescence microscope [5]. As shown in Fig. 1C, 36% of cells transfected with a plasmid carrying the full-length Dbok cDNA became apoptotic, whereas the expression of Bax induced 38% of apoptosis. Furthermore most of the Dbok-transfected cells could be rescued by co-expression of Bcl-xL or BFL-1. The N-terminaltruncated Dbok construct, Dbok-N, in which the ¢rst BH4 domain has been deleted, did not lose the apoptotic activity in REF52 cells, indicating that the N-terminal region is not necessary for its apoptotic function. One possible role of this region is not directly involved in the cell killing but rather in mediating the conformation of the protein through apoptosis stimuli. However, the C-terminal-truncated construct, Dbok-C, which lacks a putative transmembrane domain and the Dbok-BH3, which is a BH3 domain deletion construct, lost their killing activity. These results are consistent with the previous observations that mutants of the Bcl-2-related proteins lacking the transmembrane domain in the C-terminus are functionally ine¡ective. The loss of function of the C-terminal mutant indicated that the localization of Dbok is important for its function. Thus, Dbok is a pro-apoptotic protein which functions through the BH3 domain. The Cterminal sequence and the BH3 domain of Dbok are indispensable for the pro-apoptotic function but the N-terminal sequence is not required for its apoptotic potential. At this stage we do not know the function of Dbx yet. The amino acid sequence of Dbx is very similar to that of Dbok and Dbx also contains the BH4 and BH3 domains. Most interestingly a proline residue is present in the middle of the BH3 domain, which may well change conformationally the local helical structure of the BH3 domain based on the structural analysis of the Bcl-xL and B£-1 [5]. Further experiments are necessary to determine the function of this protein. Like Bok proteins from Drosophila, rat and human, chicken Bok may well function as a pro-apoptotic protein. Most recently, two Bcl-2 homology proteins have been isolated from two di¡erent species of sponges [6]. Here we report the identi¢cation of three Bok protein genes from Drosophila, human and chicken. These results indicate that the apoptosis pathway is evolutionarily conserved even from sponges to human and the complexity of regulation of apoptosis ¢ts to the evolutionary relationships among animals.

Low Multiple Pregnancy Rates and Reduced Frequency of Cancellation After Ovulation Induction with Gonadotropins, If Eventual Supernumerary Follicles Are Aspirated to Prevent Polyovulation

Purpose:Our purpose was to demonstrate the feasibility of the routine aspiration of supernumerary follicles in infertile patients with imminent polyovulation after ovulation induction with gonadotropins and to examine its effect on the frequency of cycle cancellation and on the (multiple) pregnancy rate.Methods:The data on 796 treatment cycles, performed between 1989 and 1996 on 410 infertile couples, were analyzed retrospectively. From October 1992, whenever necessary, supernumerary ovarian follicles were selectively aspirated transvaginally under ultrasound guidance to prevent the ovulation of more than three follicles. Thereafter, intrauterine insemination was performed.Results:After the adoption of transvaginal ultrasound-guided aspiration of supernumerary follicles into the treatment protocol in October 1992, the number of canceled cycles (P < 0.0001) and the multiple pregnancy rate (P < 0.01) were significantly reduced compared to those previously. The overall pregnancy rate remained stable. No ovarian hyperstimulation syndrome requiring hospitalization was noted, and no complications resulting from the follicle aspiration were registered.Conclusions:Transvaginal ultrasound-guided aspiration of supernumerary ovarian follicles increases both the efficacy and the safety of ovulation induction with gonadotropins. Because of the limited equipment required, this method represents an alternative for conversion of overstimulated cycles to more costly alternatives such as in vitro fertilization.

Production of reactive oxygen species by and hydrogen peroxide scavenging activity of spermatozoa in an IVF program

AbstractPurpose: Reactive oxygen species (ROS) including H2O2 produced by spermatozoa have been suggested, on one hand, to be associated with idiopathic male infertility and, on the other hand, to stimulate certain sperm function leading to fertilization. The influence of ROS on fertilization was investigated in 75 IVF patients by correlating fertilization rates with the production of ROS and the H2O2-scavenging activity of swim-up spermatozoa prepared in parallel with the IVF samples. Results: Low rates of ROS production by the swim-up sperm was detected by the luminol-dependent chemiluminescence assay. They were not correlated with fertilization rates. The hydrogen peroxide scavenging capacity of these spermatozoa, measured as the removal of exogenous H2O2 assayed spectrophotometrically, decreased stepwise in groups of patients achieving higher fertilization rates, suggesting a positive effect of this ROS on fertilization. An alternative explanation of this correlation is plausible in view of the association of both high scavenging activities and poor fertilization rates with poor sperm morphology. Conclusions: ROS produced by spermatozoa selected by swim-up plays no negative, if not a positive, role in fertilization.

Evaluation of in vitro cultured rat oocytes, from different strains, by spindle morphology and maturation-promoting-factor activity combined with nuclear-transfer experiments

Although successful nuclear transfer (NT) has been reported in the rat 6 years ago, somatic cell nuclear transfer (SCNT) in the rat could not be repeated. Our experiments with rat SCNT reveal the difficulties related to rat cloning. We first focussed on the most appropriate rat strain that could be used as an oocyte donor. Then we describe how rat oocytes can be kept in a nonactivated state during in vitro culture, because the latter undergo spontaneous partial activation through rapid extrusion of the second polar body after isolation from the oviduct. In the SCNT experiments performed with the one-step manipulation technique it was possible to produce rat embryos, which developed in vivo up to the blastocyst stage. In addition, we identified the implantation sites of SCNT rat embryos reconstructed with Sprague-Dawley (SD) oocytes. Furthermore, different rat strains were used as oocyte donors and their oocytes were cultured under different conditions to establish a stable nonactivating oocyte culture system. The ratio of activated to nonactivated oocytes was measured by spindle-stability and maturation promoting factor (MPF) activity. These measurements indicated that a substrain of the SD rat strain, the so-called OFA-SD strain, is the one providing the most stable oocytes, when their oocytes are cultured in the presence of the proteasome inhibitor MG132. However, it was not possible to obtain any implantation sites with reconstructed oocytes derived from the OFA-SD strain transferred to foster mothers. This goal was not achieved, even when the trichostatin A (TSA) treatment was used, which is known to enhance the cloning efficiency of reconstructed mouse, porcine, bovine, and rabbit oocytes both in vitro and in vivo by enhancing the reprogramming efficiency of the recipient nucleus.

Similar prevalence of expanded CGG repeat lengths in the fragile X mental retardation I gene among infertile women and among women with proven fertility: a prospective study

Purpose:We sought to determine the usefulness of fragile X mental retardation 1 (FMR1) carrier testing among young infertile women with or without signs of ovarian insufficiency as compared with fertile women.Methods:Three cohorts of women were recruited to determine the cytosine–guanine–guanine (CGG) repeats trinucleotide repeat length in the 5′-untranslated region of the FMR1 gene in lymphocyte DNA. A total of 199 fertile women, who were reported to have conceived within 3 months, were recruited together with 372 infertile women with ongoing menstrual cycles and 48 infertile women with primary ovarian insufficiency. The various ranges of FMR1 CGG repeat lengths among infertile women were compared with those of fertile controls. In infertile women with ongoing menstrual cycles, the serum concentrations of follicle-stimulating hormone, anti-Muellerian hormone, and inhibin B were measured during the early follicular phase.Results:None of the three categories of FMR1 CGG repeat length expansions (premutation, intermediate range, and high normal range) were more prevalent among infertile women than among fertile women. The CGG repeat length was not correlated with any of the ovarian reserve parameters.Conclusion:In comparison with a generalized preconception screening strategy, infertility as a criterion, even together with reduced ovarian reserve, is not suitable for identifying a higher proportion of women with expanded FMR1 CGG repeat length.Genet Med 16 5, 374–378.

Feasibility of dynamic MR-hysterosalpingography for the diagnostic work-up of infertile women

Background: Tubal disturbances often contribute to infertility. Conventional hysterosalpingography (HSG) is considered as standard in the assessment of the patency of the fallopian tubes, but requires ionizing radiation and is restricted to the imaging of endoluminal structures. Purpose: To evaluate dynamic magnetic resonance-HSG (dMR-HSG) in the diagnostic work-up in patients with infertility. Material and Methods: Thirty-seven consecutive infertile women underwent dMR-HSG: 20 ml of gadolinium-polyvidone solution (18.4 mM Dotarem 1:20 with polyvidone) were injected intracervically through a 5-Charriere balloon catheter while acquiring five consecutive flash-3D T1-weighted MR sequences with fat saturation. Two experienced readers assessed image quality and anatomic–pathologic correlations prospectively. The relevance of results was evaluated in the clinical context of each patient. Patient comfort was evaluated with a standardized questionnaire. Results: dMR-HSG was successfully completed in 33/37 patient with an average study time of 45 min. In 4 of 37 patients the catheter became dislodged during the examination, resulting in two complete diagnostic failures. Failure in another two patients was due to preliminary termination because of excessive pain and discomfort during the application of the contrast solution. The uterine cavity was completely visualized and bilateral fallopian tube patency was confirmed by dMR-HSG in 27 of 33 patients. Bilateral tubal occlusion was diagnosed in one of the remaining six patients and was confirmed by laparoscopy. Successful selective tubal catheterization was performed in one additional patient with unilateral and one patient with bilateral fallopian tube occlusion. In three cases, the catheter became dislocated at the end of the examination without demonstration of tubal patency. Since all three patients refused diagnostic laparoscopy and conventional HSG, possible bilateral occlusions of the fallopian tubes could not be further assessed. Conclusion: dMR-HSG with cervical cannulation and intracavitary gadolinium injection is feasible and allows assessment of the uterus, the fallopian tubes, and extra-uterine pelvic structures, while avoiding all ionizing radiation in infertile women aiming at pregnancy.