Genomic DNA in human blastocoele fluid.

IVF often requires embryo cryopreservation through vitrification. During the vitrification process, the embryos can be collapsed by withdrawing the blastocoele fluid. The metabolomic profile of blastocoele fluid has been recently investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry to provide metabolite information that can help estimations of implantation efficiency. However, the presence of embryo DNA in blastocoele fluid has not been reported to date. This study shows using real-time PCR that genomic DNA was present in about 90% of blastocoele fluid samples harvested during the vitrification procedure. Moreover, the potential for determining embryo sex directly from blastocoele fluid is demonstrated by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). This opens up the possibility of screening embryos from couples carrying an X-linked disorder to identify male embryos at high risk of disease. The application of whole-genome amplification technologies to fluid samples is also shown to be feasible, potentially allowing more comprehensive genetic tests. As proof of principle, microarray comparative genomic hybridization was attempted to confirm the sex of embryos as well as detect several aneuploidies. However, further studies are needed to validate this approach and confirm that the accuracy is sufficient for diagnostic purposes.

Treatment strategies of the borderline ovarian tumors.

The present study was undertaken to establish the role of surgical procedures, histologic type, and stage of the tumor on the survival rate of patients with borderline ovarian tumors in a 5 to 15 years of follow-up. Data reported in the literature have shown the low malignancy of this cancer and that only the stage, but not the pathological diagnosis, is significantly influencing the survival rate of the patients. After 5 years, the survival rate of patients with tumors of stage I to stage II is 98.2% (n=567) and 81.4% (n=46), respectively, with no statistical difference. After 5 years, survival rate between tumors of stage I to stage III is 98.2% (n=567) and 79.1% (n=96), respectively (p< 0.05). The data shows that for borderline ovarian tumors, a minimally invasive surgery is warranted.

The impact of embryo transfer on implantation--a review.

Embryo transfer has received little clinical attention and has been, until recently, the most inefficient step in in-vitro fertilization (IVF). In this article, the authors review the literature and their personal experience regarding the process of intrauterine transfer of embryos, which remains the object of much discussion. Factors which appear to influence implantation rates are: contamination of the catheter tip with cervical bacteria, stimulation of uterine contractions during the procedure, the type of catheter, ultrasound guidance during the transfer, and the position of the embryos in the uterine cavity. Easy and atraumatic transfer is essential for successful implantation and the embryos need to be placed in the middle of the cavity, away from the fundus. Knowing, beforehand, the position and length of the uterus can provide better results and may reduce the rate of ectopic pregnancies. Evidence from randomized studies has supported this claim. Despite the number of available studies controlling certain variables, most authors, even using the same catheter, ultrasound guidance and/or a trial transfer use different protocols or similar instruments in different ways. Standardization of the transcervical intrauterine transfer of embryos in a large randomized study is needed before definitive conclusions can be drawn. The goal of improved implantation and pregnancy rates deserve these efforts.

Paracrine regulation of menstruation.

Endometrial proliferation, secretion, vascular neoformation and modification to shedding is under direct and/or indirect control of steroid hormones. The progressive modification of the endometrial architecture is due to its growth and differentiation. The new tissue regenerates monthly from a 2-5 mm to a 12-18 mm of complex tissue until it sheds under a co-ordinated network of bioactive molecules produced and activated during the menstrual cycle. The steroid hormones, the HLA-DR and integrin molecules, the intense production of several proteins, the vascular damage, and the disconnection of cell-cell and cell-matrix interaction are participating in both the endometrial preparation for embryonic implantation and the shedding and bleeding of the tissue itself. Menstruation is a process associated with damage to the epithelium, endothelium and extracellular matrix, ending on controlled bleeding, tissue dissolution and repair. Endometrial proteinases and tissue factor (TF) contribute to systemic factors to control the mechanisms of regulation of tissue dissolution, tissue shedding, and vascular bleeding during menstruation.

The hormonal control of endometrial receptivity: estrogen (E2) and progesterone.

While the number of identified substances produced by the ovary increases steadily, it remains remarkable that the sole use of exogenous estrogen (E2) and progesterone (P) can prime optimal endometrial receptivity in women whose ovaries have failed or are absent. Early work showed that a marked leeway existed in the acceptable duration of the E2-only phase of endometrial priming. Subsequently, a sequence of transformations are induced by exogenous progesterone that reproduces classical findings made in the menstrual cycle. Secretory changes in endometrial glands are best seen between the 4th and 6th day of progesterone administration (day 18-20 of an ideal cycle where progesterone exposure starts on day 15). Predecidual changes of the endometrial stroma are apparent starting on the 10th day of progesterone exposure (day 24). Contrary to earlier belief, even maximal alterations in the plasma E2 to progesterone ratio fails to alter the endometrial morphology of either glands or stroma. More recently it has been recognized that E2 and progesterone also affect uterine contractility. It has been postulated that excessively high levels of E2 may increase uterine contractility and adversely affect implantation rates in in-vitro fertilization (IVF). Exogenous progesterone has been shown to exert utero-relaxing effects and it has been hypothesised that progesterone supplementation before embryo transfer (ET) may improve receptivity in IVF.

Immunologically mediated abortion (IMA).

Roughly 20% of all clinical pregnancies evolve into "spontaneous abortions". The causes of spontaneous abortion have been determined in under 60% of the total and comprise genetic, infectious, hormonal and immunological factors. In some cases the immune tolerance mechanism may be impaired and the foetus immunologically rejected (IMA, immunologically mediated abortion). The immunological mechanism implicated depends on the time in which pregnancy loss takes place. During preimplantation and up to the end of implantation (13th day) the cell-mediated immune mechanism (potential alloimmune etiologies) is responsible for early abortion. This mechanism involves immunocompetent decidual cells (eGL, endometrial granulated lymphocytes) already present during pre-decidualization (late luteal phase) and their production of soluble factors or cytokines. Once the implantation process is over, after blastocyst penetration of the stroma and the decidual reaction of uterine tissue, IMA could be caused by cell-mediated and humoral mechanism (anti-paternal cytotoxic antibodies or autoantibody etiology), by the production of paternal anti major histocompatibility complex antibodies, or even by an autoimmune disorder leading to the production of autoantibodies (antiphospholipid antibodies, antinuclear antibodies or polyclonal B cell activation). The diagnostic work-up adopted to select IMA patients is crucial and includes primary (karyotype of both partners, toxo-test, hysterosalpingography, endometrial biopsy, thyroid function tests, serum hprolactin, luteal phase dating) and secondary (full hemochromocytometric test, search for LE cells, lupus anticoagulant, anticardiolipin, antinuclear antibodies, Rheumatoid factor, blood complement VDRL) investigations. Therapeutical approaches vary. If autoimmune disorders are demonstrated therapies with different combinations of corticosteroids, aspirin and heparin or intravenous immunoglobulin are administered. Otherwise, therapy with paternal or donor peripheral blood mononuclear cells should be instituted.

Dysfunctional uterine bleeding (DUB).

Cyclic or irregular uterine bleeding is common in perimenarchal and perimenopausal women with or without endometrial hyperplasia. The disturbance often requires surgical treatment because of its negative effects on both blood loss and abnormal endometrial growth including the development of endometrial cancer. The endometrium is often overstimulated during the perimenopausal period when estrogen/progesterone production is unbalanced. A therapeutical approach with gonadotropin-releasing hormone agonist (GnRHa) was proposed in a depot formulation (Zoladex) that induces a sustained and reversible ovarian suppression. To avoid the risk of osteoporosis and to obtain adequate endometrial proliferation and differentiation during ovarian suppression, transdermal 17-beta-estradiol and oral progestin were administered. Results of 20 cases versus 20 controls showed a reduction of metrorrhagia, a normalization of hemoglobin plasma concentration, and an adequate proliferation and secretory differentiation of the endometrium of patients with abnormal endometrial growth. Abnormal uterine bleeding is mainly due to uterine fibrosis and an inadequate estrogen and/or progesterone production or to a disordered estrogen transport from blood into the endometrium. In premenopausal women, endometrial hyperplasia may be part of a continuum that is ultimately manifested in the histological and biological pattern of endometrial carcinoma. The regression of endometrial hyperplasia obtained by using the therapeutic regimen mentioned above represents a preventive measure for endometrial cancer. Finally the normalization of blood loss offers a good medical alternative to surgery for patients with DUB.

The first uterine pass effect.

The endometrial effects of vaginal progesterone have been found to be unexpectedly reliable. This has led us to suspect that a local direct vagina-to-uterus transport or first uterine pass effect was the basis of the uterine targeting of vaginal progesterone. After vaginal administration of progesterone, uterine tissue concentration has been found to exceed by more than 10-fold the levels achieved by systemic administration, despite plasma levels in the latter case that were more than seven times higher. Similar differences in systemic-to-uterine tissue level ratios have been observed between oral and vaginal administration of danazol. Originally seen as a pharmacological advantage permitting the uterine targeting of vaginally administered substances, it is possible that the first uterine pass effect plays a physiological role in the control of uterine contractile activity through the prostaglandins contained in the semen.

Tissue factors influencing growth and maintenance of endometriosis.

The unpredictable response of endometriosis to steroids and its recurrence after therapy, led us to hypothesize a possible further control of this pathology by factors other than steroids. The presence of estrogen, progesterone and epidermal growth factor receptors (ER, PR, EGFr) was evaluated using immunohistochemistry before and after therapy with Danazol or a gonadotropin-releasing hormone analogue (GnRHa), Buserelin. EGFr, ER and PR were present in 100% of endometrial specimens, and in 71%, 29% and 49% of endometriotic implants, respectively. Danazol and GnRHa reduced immunohistochemical staining for EGFr antisera in the endometrial and endometriotic specimens. About 21% of endometriosis were EGFr positive and ER negative, suggesting a potential role of epidermal growth factor in growth and maintenance of endometrial ectopia.

The effect of progestin on factors influencing growth and invasion of endometrial carcinoma.

Progesterone (P) and progestins play an important role in the control of endometrial growth. We have investigated P and progestin effects on endometrial estrogen extraction, on basement membrane (BM) synthesis and on the presence of the epidermal growth factor receptor (EGFr) in normal and pathologic endometrium. E2 uptake, evaluated in human isolated perfused uteri is significantly decreased by P. BMs investigated using immunohistochemistry, with antisera to collagen IV and laminin, were found around stromal cells only in the luteal phase or during P or progestin administration. Glandular BM, discontinuous in hyperplastic and carcinomatous endometria, were restored to integrity only in typical hyperplasia after therapy with progestin. Endometrial EGFr is modified by P: revelation of this antigen is increased in proliferative phase and decreased in secretory phase. Similarly this molecule was present in hyperplastic and carcinomatous endometria. Only in benign hyperplasia did we observe no staining for the same antigen after progestinic therapy. These data suggest that P or progestins may also have an indirect influence through mechanisms such as estrogen uptake and tissue factor activity with important differences between normal and pathologic endometrium.

Implantation failure in assisted reproduction technology and a critical approach to treatment.

In this article, we review the literature and our personal experience regarding the many factors that appear to influence implantation rate. Oocyte quality, as determined by patient age and aneuploidies, probably plays a major role in RIF. However, a panoply of other factors have been brought under investigation, quite often with contradictory results and additional intriguing questions to be studied. Infections of the vagina, cervix, and endometrium, the role of mucus aspiration and washing of the cervix on transfer, the role of catheter guidance for a correct transfer and potion of embryos, the effect of mock transfer, and the role of hysteroscopy and its timing before transfer procedures are analyzed both as a review of the literature and as opinions and data from our experience. Many of these factors are interlaced and from the apparently simple issue of trauma, to infections and immune modulation of hatching and implantation, a biological continuum can easily be identified. The impact of abnormalities of the immune system and of homeostasis abnormalities is also covered in a brief overview of reported works and our experience. These latter areas probably constitute the common biological background of all other external factors that, however, the skilled must equip themselves for improving implantation success.

Effects of vaginal progesterone on pain and uterine contractility in patients with threatened abortion before twelve weeks of pregnancy.

Fifty women with previous diagnosis of inadequate luteal phase and threatened abortion underwent a prospective, randomized, double-blind study in one medical center carried out with a parallel trial. The primary objective was to establish the effects of vaginal progesterone (Crinone 8%) in reducing both pain and uterine contractions (UCs). The gel with or without (placebo) vaginal progesterone was administered once a day since the diagnosis of threatened abortion and for 5 days. The efficacy on pain symptom amelioration was evaluated by a 5-score intensity gradation, while the UCs were evaluated by ultrasound. The secondary objective of the study was to evaluate the outcome of the pregnancies. The use of progesterone was effective both on pain relief and on the frequency of the UCs that decreased after 5 days of vaginal progesterone administration (P < 0.005). The evaluation of the ongoing pregnancy and spontaneous abortion in both study groups after 60 days showed that 4 patients of group A and 8 patients of group B miscarried (P < 0.05). In conclusion, patients with threatened abortion benefit from vaginal progesterone by a reduction of UCs and pain. The use of vaginal progesterone improved the outcome of pregnancies complicated by threatened abortion and previous diagnosis of inadequate luteal phase.

Basement membrane in human endometrium: possible role of proteolytic enzymes in developing hyperplasia and carcinoma.

Basement membranes (BM) are elements of the extracellular matrix that are essential for growth and differentiation of tissues. Several collagenolytic enzymes of tumor cells are involved in degradation of the extracellular matrix; growth and inhibitor factors [e.g. Epidermal Growth Factor (EGF), Transforming Growth Factors alpha and beta (TGF-alpha, beta)] seem to be involved in the extracellular matrix formation and degradation. To establish a possible association between the presence of collagenase (C), urokinase-type plasminogen activator (uPA) and the neoplastic growth of the endometrium, 44 endometrial specimens (14 proliferative, 11 secretive, 7 adenomatous hyperplasia, 12 adenocarcinoma) were studied using immunohistochemistry with antisera for C, uPA, EGF receptors and TGF-alpha. Immunostaining for collagenase revealed a positive reaction in moderately differentiated adeno-carcinoma without staining the normal and hyperplastic endometrium. A progressive increase in uPA immunostaining was observed in proliferative and neoplastic endometrium. TGF-alpha and its receptor (EGFr) were stained in proliferative and more clearly in hyperplastic and carcinomatous endometrium. In conclusion, BM play an important role in proliferation and differentiation of human endometrium; their degradation influences estrogen transportation from blood to the stroma. Endometrial BM degradation is associated with the presence of collagenolytic enzymes and growth factors.

Uterine contractility: vaginal administration of the beta-adrenergic agonist, terbutaline. Evidence of direct vagina-to-uterus transport.

Spontaneous uterine contractility during the menstrual cycle is required for menstruation, gamete transport, and, most likely, embryo nidation. Abnormal uterine contractility has been linked to dysmenorrhea, a condition associated with painful uterine cramping. Based on previous studies with progesterone, we have postulated the existence of a portal system that is responsible for some degree of direct vagina-to-uterus transport of administered compounds (i.e., the "first uterine pass effect"). It is possible that treatment with uterorelaxing substances, particularly beta-adrenergic agonists, may alleviate the uterine discomfort that accompanies dysmenorrhea. However, side effects encountered with oral administration of beta-agonists limit their utility. Alternatively, vaginal delivery of beta-agonists could solve this dilemma by enhancing their efficacy and reducing side effects. Therefore, in the current study we used hysterectomy specimens and an in vitro uterine perfusion system to test the vagina-to-uterus transport of [3H]terbutaline, a well-known beta-agonist. With the use of autoradiographic and scintillation counting techniques, our results clearly show progressive diffusion of labeled terbutaline from the rim of vaginal tissue through the uterus during the first 12 hours of perfusion. This indicates that uterine targeting of terbutaline can be accomplished through vaginal administration, suggesting a new therapeutic modality in women's health care.

Contractility of the nonpregnant uterus: the follicular phase.

Recent renewed interest in uterine contractility stems from the possibility of directly visualizing uterine contractility on images generated by high-resolution ultrasound probes. During the menstrual cycle, three typical patterns of uterine contractility have been recognized. During the luteofollicular transition and early follicular phase (menses), the contractile event involves all layers of the myometrium and exerts antegrade (from fundus to cervix) expulsive forces. Characteristically, uterine contractions are often perceived by women at the time of menses, sometimes reaching the level of painful cramps (dysmenorrhea). In the late follicular phase, uterine contractility involves only the subendometrial layers of the myometrium and is never perceived by women. The primary function of uterine contractility in the late follicular phase is to facilitate the retrograde (cervix to fundus) transport of sperm towards the distal end of the fallopian tubes where fertilization normally takes place. Finally, the uterus reaches a stage of quiescence after ovulation (under the influence of progesterone) that characterizes the major part of the luteal phase. The present review summarizes our understanding of the physiological role of uterine contractility during the follicular phase and the possible implications in pathological circumstances such as endometriosis and dysmenorrhea.

Factors regulating interaction between trophoblast and human endometrium.

Implantation is a crucial step in human reproduction. Disturbances of this process are responsible for pregnancy failure after both in vivo and in vitro fertilization. The endometrium provides the implanting embryo with a unique substratum where the embryo communicates with biochemical signals, attaches itself, penetrates and grows without blood circulation. The highly proliferative phase of the cytotrophoblast, during early human embryogenesis, may be due to endogenous production of growth factors that may establish autocrine/short range paracrine stimulator loops which explain the tumor-like properties of these tissues. Endometrial BM penetration and stroma invasion may be due to the proteolytic capability of the human embryo. It is suggested that collagenase and the urokinase-like plasminogen activator are responsible for this activity. To clarify the molecular mechanisms involved in human embryo implantation several models are suggested: culture of blastocysts, culture of endometrial cells, and endometrial explant co-culture. Human blastocysts cultured with whole perfused human uteri make it possible to recognize some aspects of the entire implantation process and give us the possibility of improving the benefits provided by new technologies in reproductive medicine and reducing embryonic loss at an early stage.

Direct transport of progesterone from vagina to uterus.

OBJECTIVE: To compare progesterone concentrations in serum and endometrial tissue from hysterectomy specimens after vaginal or intramuscular (IM) administration of progesterone gel. METHODS: This was a randomized open study of 14 post-menopausal women undergoing transabdominal hysterectomies. Participants received either vaginal progesterone gel, 90 mg, or IM progesterone, 50 mg, at 8:00 AM and 8:00 PM on the day before surgery and at 6:00 AM on the day of surgery. Venous blood samples for progesterone measurement were collected at 8:00 AM on the day before surgery (baseline) and during surgery. After removal of the uterus, the endometrium was sampled from the anterior and posterior walls. Results were expressed as ratios of endometrial to serum progesterone concentrations x 100. RESULTS: Ratios of endometrial to serum progesterone concentrations were markedly higher in women who received vaginal progesterone (14.1 median, 8.5-59.4 range; 95% confidence interval [CI] 9.89, 38.79) compared with IM injections (1.2 median, 0.5-13.1 range; 95% CI -0.48, 7.39) (P < .005). CONCLUSION: Ratios of endometrial to serum progesterone concentrations were higher after vaginal administration of progesterone than after IM injections. Our findings in endometrial tissue specimens from hysterectomies excluded the possibility of contamination by progesterone that remained in the vagina.

Dating the endometrial biopsy by flow cytometry.

OBJECTIVE: To determine endometrial changes throughout the menstrual cycle. DESIGN: Flow cytometric analysis of endometrial cells versus chronological dating. SETTING: Women volunteers with a normal menstrual cycle participated in this study that was done in an academic research environment. PATIENTS: Two hundred thirty regular menstruating women with adequate luteal phase underwent endometrial biopsy at different days of their menstrual cycle; 138 biopsies were analyzed both histologically and by flow cytometry. MAIN OUTCOME MEASURE: Percentage of biopsies correctly classified in terms of chronological dating. RESULTS: Flow cytometry correctly classified the chronological day of biopsies in 59% of cases, the proliferative versus secretory days in 91% of cases, and the early, mid, and late secretory phases of the menstrual cycle were correctly predicted in 86% of cases. CONCLUSION: Flow cytometry is a good method to determine growth and differentiation of the endometrium during the menstrual cycle. Detection of phases of the whole endometrial cell population offer information on the biological adequacy of endometrial response to the biochemical environment.

A 48-hour preservation of an isolated human uterus: endometrial responses to sex steroids.

Human uteri were perfused with Krebs-Ringer bicarbonate-glucose buffer with and without estrogens and progesterone for a period of up to 48 hours to preserve a viable organ, which was responsive to hormones. Flow rates of 12 to 35 ml/minute per artery were fully distributed into the organ, with pressure values ranging from 80 to 120 mm Hg. Arteriovenous gradients of oxygen and carbon dioxide tensions as well as the levels of lactate, lactic dehydrogenase, and creatine kinase released in the perfusate, indicators of tissue ischemia or cell necrosis, showed a good preservation of the organ for up to 48 hours. The light- and electron-microscopic examinations of endometrial and myometrial tissues taken before and during perfusion confirmed this result. The extracorporeal perfusion of uteri with buffer containing estrogens plus progesterone exhibited secretive modifications of the proliferative endometrium, thus suggesting the viability of the organ and its responsiveness to sex steroids.

Treatment of hirsutism by an association of oral cyproterone acetate and transdermal 17 beta-estradiol.

Twenty-four hirsute women were treated with an inversal sequential scheme of cyproterone acetate, 50 mg/d by oral route from the 1st to the 15th day of the menstrual cycle, along with 100 micrograms/24 h of 17 beta-estradiol transdermally administered from days 1 to 21, for nine cycles at weekly intervals. The acne and seborrhea as well as hirsutism showed a significant improvement in all subjects studied. The plasma testosterone and dehydroepiandrosterone sulfate decrease from 1.5 +/- 1.3 ng/mL and 6.9 +/- 1.3 micrograms/mL to 0.5 +/- 0.03 ng/mL and 2.7 +/- 1.7 micrograms/mL, respectively. Similar values were observed in subjects with idiopathic hirsutism during the treatment. The metabolic parameters, as well as the plasma levels of sex hormone-binding globulin, appeared unaffected by the therapy. Furthermore, the luteinizing hormone and follicle-stimulating hormone secretion was strongly inhibited from the first cycle of treatment. In conclusion, considering the good clinical results and the avoidance of any hepatic effect, this association should be taken into account in the treatment of hirsutism, especially in case of oral estrogen intolerance.