The expression of receptivity markers in the fallopian tube epithelium.

Pinopodes represent the morphological and integrins, the biomolecular markers of endometrial receptivity. We studied using scanning electron microscopy, the expression of pinopodes on tubal samples and their corresponding endometria, from 21 women of reproductive age (7 from proliferative phase, 7 from day LH +5 and 7 from day LH +7). In addition, we examined the immunohistochemical staining of integrins alpha v beta 3, alpha v beta 5 and their ligands, fibronectin (FN) and osteopontin (OPN) in the same tubal epithelium samples. Pinopodes were detected on the tubal epithelium exclusively during day LH +7, coincident with their formation in the endometrium and synchronous to alpha v beta 3 sharp increase in the oviduct epithelium, suggesting a regulation similar to the endometrium. In contrast, alpha v beta 5, FN and OPN remained unchanged during the cycle. These results show for the first time the formation of pinopodes in the tubal epithelium at the time of endometrial receptivity and correlate it with the upregulation of the intact dimmer alpha v beta 3 in the tubes.

Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure.

We have developed a coculture system with autologous human endometrial epithelial cells (AEEC) that retained many features of human endometrial epithelium. Implantation failure (IF; >3 previous cycles failed with 3-4 good quality embryos transferred) is a distressing condition in which 2-day embryo transfer repetition is the routine option. The objective of this study was to investigate the basics and to evaluate prospectively the clinical value of embryo coculture on AEEC and blastocyst transfer with their own oocytes [in vitro fertilization (IVF) patients] or with donated oocytes (oocyte donation patients) compared to a routine day 2 embryo transfer for patients with IF. Scanning electron microscopy and mouse embryo assays demonstrate that EEC from fertile and IF patients were morphologically and functionally similar; similar findings were observed in EEC obtained from fresh or frozen endometria. Clinically, 168 IVF cycles were performed in 127 patients with 3.8+/-0.2 previously failed cycles, and 80 cycles were performed in 57 patients undergoing oocyte donation with 3.0+/-0.2 previously failed cycles. Twenty IVF patients and 15 ovum donation patients with 3 previously failed cycles in whom a 2-day embryo transfer was performed were used as controls. In 88% of ovum donation cycles, at least 2 blastocysts were available for transfer, with 60.1% blastocyst formation; 2.2+/-0.1 blastocysts were transferred/cycle, and 36 pregnancies (determined by fetal cardiac activity) were obtained (32.7% implantation and 54.5% pregnancy rates). In 168 IVF cycles, 8.1+/-0.2 embryos/cycle started coculture, resulting in 49.2% blastocyst formation; 2.3+/-0.2 blastocysts were transferred/cycle, and 29 clinical pregnancies were obtained (11.8% implantation and 20.2% pregnancy rates). Fifteen cycles were canceled (9%). In oocyte donation patients with IF undergoing 2-day embryo transfer, implantation and pregnancy rates were significantly lower (4.5% and 13.3%; P < 0.01) than with coculture; however, in IVF patients with IF, results with day 2 transfer (10.7% and 35%) were similar to those with coculture. The present study demonstrates that coculture of human embryos with AEEC and blastocyst transfer is safe, ethical, and effective and constitutes a new approach to improve implantation in patients with IF undergoing ovum donation, but not in IVF patients.

Uterine pinopodes in peri-implantation human endometrium. Clinical relevance.

Current clinical studies indicate the existence in the human of an "implantation/nidation window," similar to that observed in animal models. During this short period of uterine receptivity, the formation of pinopodes on the apical cell membrane of the endometrial epithelium is a consistent morphological event, observed in a number of species studies including the human. In order to develop a specific marker of the implantation window in clinical practice, we have investigated the kinetics of pinopode formation through sequential endometrial sampling under various hormonal conditions. Our results show that the implantation window in humans, according to this marker, lasts less than 48 hours, and the timing of its opening is dependent on the hormonal treatment applied, occurring earlier in cycles following ovarian stimulation and later in cycles induced by hormone replacement treatment. Furthermore, the timing varies among different individuals under the same treatment. These findings suggest that examination for pinopodes in endometrial samples can be highly useful in infertility treatment and research for the assessment of the nidation window on an individual basis. Our preliminary data strongly support the value of this assessment for better timing of ovum transfer, leading to an increase in implantation rates. Studies are now in progress on the expression of other endometrial signals present in relation to the pinopodes.

Surface morphology of the human endometrium. Basic and clinical aspects.

The human endometrium is an extremely sensitive target for steroid hormones. During the menstrual cycle, this tissue undergoes dynamic changes that are reflected on the surface morphology of the epithelium and that can be followed by scanning electron microscopy. The morphologic changes peak at the midsecretory phase, with the formation of the so-called pinopodes. Increasing evidence suggests that these pinopodes are accurate markers for endometrial receptivity, and their detection may be of high clinical utility in the preparation of endometrium before embryo transfer. This article recapitulates published figures of endometrial ultrastructure and presents some unpublished data from ongoing studies.

Endometrial receptivity: changes in cell-surface morphology.

Ovulation and fertilization trigger embryonic development and endometrial differentiation by corpus luteum progesterone production. These two synchronous processes couple about 1 week later, when the blastocyst begins to implant in the now receptive endometrium (implantation window). Receptivity is a state of endometrial differentiation marked by a change in epithelial morphology: the hairy-like cell microvilli fuse to a single flower-like membrane projection called the "pinopode." Scanning electron microscopy of sequential endometrial biopsies shows that pinopodes form briefly (1-2 days), and their numbers correlate with implantation. On average, the formation of pinopodes is earlier in stimulated (days 19-20) and later in artificial (days 21-22) compared with natural cycles (days 20-21). There is, however, a wide (up to 5 days) variation between women in the cycle days on which pinopodes form. These results suggest the existence of a narrow and discrete implantation window in humans. Detection of pinopodes is a potential clinical marker to assess endometrial receptivity.

Detection of endometrial pinopodes by light microscopy.

OBJECTIVE: To study the value of light microscopy (LM) in the assessment of endometrial pinopodes. DESIGN: Comparative histologic study. SETTING: Outpatient infertility clinic in an academic teaching institution. PATIENT(S): Eighteen oocyte donors undergoing controlled ovarian hyperstimulation. INTERVENTION(S): Endometrial biopsies on days 14-24 of the cycle. MAIN OUTCOME MEASURE(S): Assessment of pinopodes by scanning electron microscopy (SEM) and of endometrial surface projections by LM. RESULT(S): The luminal surface was identified by LM in 36 of 38 endometrial specimens obtained. Although apical projections could be recognized in all, they were few, moderate, and abundant in 20, 12, and 4 cases, respectively. Pinopodes were detected by SEM in all 4 samples with abundant projections, but in only 14 of 32 samples with lesser quantities of these surface features. No predictive value could be ascribed to apical projections viewed by LM for the developmental stage of pinopodes as defined by SEM. CONCLUSION(S): The LM of routine endometrial specimens can serve as a preliminary tool in the evaluation of surface morphology. Although abundant apical projections by LM are compatible with the presence of pinopodes by SEM, the latter modality remains as the definitive method in cases with few or moderate projections and for the evaluation of the stage of pinopode development.

Endometrial estrogen and progesterone receptor and pinopode expression in stimulated cycles of oocyte donors.

OBJECTIVE: To study the effect of controlled ovarian hyperstimulation and the ovarian response on several features of endometrial morphology simultaneously. DESIGN: Prospective controlled study. SETTING: Academic infertility center. PATIENT(S): Twenty-five oocyte donors undergoing COH and 10 ovulatory controls. INTERVENTION(S): Endometrial biopsies during the luteal phase and measurement of serum E2 and progesterone levels on days 12, 13, and 18-20. MAIN OUTCOME MEASURE(S): Endometrial morphology as judged by histologic dating, pinopode expression, and estrogen and progesterone receptor content. RESULT(S): Controlled ovarian hyperstimulation caused the early expression of endometrial features as judged by histologic dating criteria, estrogen and progesterone receptor expression, and the timing of pinopode expression in many of the subjects. A significant correlation within subjects with regard to their particular result on any one measure (e.g., histologic examination) and the others (e.g., estrogen and progesterone receptors, pinopodes) was observed. Those with higher levels of progesterone the day after hCG administration exhibited the most prematurity of morphologic features. CONCLUSION(S): Many controlled ovarian hyperstimulation cycles are associated with synchronous early expression of the expected pattern of histologic features, estrogen and progesterone receptors, and pinopodes. The most predictive feature of this premature expression was the level of progesterone the day after hCG administration.

Formation of pinopodes in human endometrium is associated with the concentrations of progesterone and progesterone receptors.

OBJECTIVE: To investigate the relation between the development of endometrial pinopodes and the serum concentration of hormones and the distribution of estrogen receptor-alpha, estrogen receptor-beta, progesterone receptor A, and progesterone receptor B. DESIGN: Prospective clinical study. SETTING: Hospital-based unit of reproductive health and university-affiliated reproductive research laboratories. PATIENT(S): Twenty-seven healthy fertile women with normal menstrual cycles. INTERVENTION(S): Urine and blood sampling for hormone measurement, vaginal ultrasonography, and endometrial biopsy. MAIN OUTCOME MEASURE(S): Appearance of the endometrium on light microscopy, pinopode formation, serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH), and expression of progesterone receptors A and B and estrogen receptors alpha and beta. RESULT(S): Pinopode formation and regression were closely associated with increases and decreases, respectively, in serum progesterone concentration. At pinopode development, levels progesterone receptors A and B in the glandular and luminal epithelial cells decreased; this effect was mainly dependent on the absence of progesterone receptor B. Serum estrogen levels and levels of estrogen receptor alpha and beta did not correlate with pinopode formation. CONCLUSION(S): The increase in serum progesterone level and down-regulation of progesterone receptor B are important in development of pinopodes.

Endometrial receptivity in terms of pinopode expression is not impaired in women with endometriosis in artificially prepared cycles.

OBJECTIVE: To assess endometrial receptivity in terms of pinopode expression in women with endometriosis. DESIGN: Prospective, observational study. SETTING: Oocyte donation program at the Instituto Valenciano de Infertilidad. PATIENT(S): Twelve women with endometriosis as the only cause of infertility. INTERVENTION(S): Pinopode expression pattern analysis by scanning electron microscopy in two sequential endometrial biopsies obtained in the same cycle of each patient. MAIN OUTCOME MEASURE(S): Pinopode pattern and pregnancy rates. RESULT(S): Pinopode expression in women with endometriosis did not differ from that of patients without endometriosis undergoing artificial cycles. Similarly, the clinical outcome in these women was comparable to that of the general population included in the oocyte donation program. The pregnancy rate per transfer was 46.7%. CONCLUSION(S): These results show that in women with endometriosis undergoing oocyte donation under hormone replacement therapy, pinopode expression is not altered, suggesting that endometrial receptivity in women with this disease remains unaltered.

In vivo phytochrome-mediated perception of reflected light signals.

The spectrophotometric assay of phytochrome in vivo in etiolated plant material was used to determine the effects of changes in reflected light on the state of the photoreceptor in etiolated seedlings exposed simultaneously to direct and reflected light. Changes in reflected light that were small in terms of the total (direct + reflected) radiation incident on the seedlings produced detectable changes in the state of phytochrome in vivo. The contribution of reflected light to the state of phytochrome in vertical organs was greater than expected from its low contribution to total incident light. These data from laboratory studies complement and are consistent with results of field studies on the effects of light reflected from neighboring vegetation on plant growth under natural radiation conditions.

Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus.

We have shown previously that the epithelial cells of human endometrium produce CRH. The biological role of endometrial CRH is not yet known. Among other things, CRH appears to be involved in the inflammatory process, acting as an autocrine/paracrine proinflammatory regulator. Since the reaction of endometrium to the invading blastocyst has characteristics of an aseptic inflammatory reaction, we have hypothesized that endometrial CRH may participate in the inflammatory phenomena taking place at the implantation site of blastocyst. In the present study we demonstrate a higher content of immunoreactive (IR)-CRH and CRH mRNA in the implantation sites of early pregnant rat uterus compared to the inter-implantation regions. Specifically we have found that: a) rat uterus contained a 1.3 kb CRH transcript, similar or identical in size to that present in human placenta, b) the size of the IR-CRH present in uterine extracts was similar to authentic hypothalamic CRH, c) Northern blot analysis showed that the content of CRH mRNA in uterus at the implantation sites was 3.5 fold higher compared to that in the inter-implantation regions and finally, d) immunohistochemical localization of IR-CRH in early pregnant rat uterus revealed positive staining of the luminal epithelial cells in both implantation and inter-implantation uterine regions, while decidualized stromal cells were positive only at the implantation sites. Our data suggest that endometrial CRH may play a role in the implantation of blastocyst.

Cell-surface morphological events relevant to human implantation.

Morphological evidence on early stages of human implantation is limited to very few sporadic observations. The nature of implantation which requires the presence of both maternal and embryonic tissues, combined with the currently existing ethical constraints on human studies, appear to preclude generation of new data. However, research on relevant animal and in-vitro models as well as studies on human endometrium and in-vitro embryos, allow some indirect insights to this phenomenon. This review summarizes information on cell-surface morphological events relevant to implantation initiation, derived from scanning electron microscopy studies on the above systems. A central part of this article deals with the formation of epithelial cell projections known as pinopodes, for there is increasing evidence suggesting that these structures are closely associated with the development of endometrial receptivity for blastocyst implantation in humans.

Pinopodes as markers of endometrial receptivity in clinical practice.

Clinical evidence indicates the existence in the human of a narrow window of uterine receptivity which opens during the mid-luteal phase. At the same time, formation of pinopodes on the apical membranes of the endometrial epithelial cells occurs. To develop a specific marker for receptivity in clinical practice, the kinetics of pinopode formation has been investigated through sequential endometrial biopsying in natural, hormone replacement and stimulated cycles. The results show that pinopodes last <48 h in all cycles, and the timing of their formation depends both on the hormonal treatment applied and on the patient's individual response. On average, pinopodes form earlier in stimulated cycles and later in hormone replacement cycles, compared with natural cycles. Pinopode expression is strongly correlated with implantation following embryo transfer and furthermore, detection of pinopodes in assessment cycles may be extremely useful for the assessment of receptivity on an individual basis to optimize embryo transfer, resulting in increased implantation rates. Finally, pinopodes seem to be correlated well with other cellular and molecular events occurring in the mid-luteal endometrium.

The role of prostaglandins in blastocyst implantation.

This review summarizes the information available on the involvement of prostaglandins in blastocyst implantation, and examines their interactions with three other inflammatory mediators, platelet-activating factor (PAF), interleukin (IL-1) and corticotrophin-releasing factor (CRF). Essential elements of this information, consistent with the assumption that prostaglandins play an important role in implantation, appear to be: (i) the burst of endometrial prostaglandin production, following the blastocyst signal(s) or an artificial stimulus; (ii) the main localization of this production at the luminal epithelium and release towards the stroma; and (iii) the presence at the stromal level of specific progesterone-dependent binding sites for prostaglandin E2. In addition, accumulated data indicate a paracrine interaction at the endometrial level between PAF and prostaglandin E2, which could serve, among others, to amplify the embryonic signal(s). Pro-inflammatory cytokines IL-1 alpha and IL-1 beta may also play a significant role in endometrial response via the modulation of prostaglandin E2 production. Prostaglandins and IL-1 induce the expression of CRF, which acts as an autocrine/paracrine inflammatory regulator. CRF exhibits a strong vasoactivity in skin tests, inducing a local increase of capillary permeability at a concentrations of 10(-10) M. Levels of CRF and its mRNA were found to be higher in rat implantation sites compared with those in the interimplantation regions. Stromal cells were found to be positive for immunoreactive CRF at the implantation sites only. It is suggested that CRF may be involved in the local increase of capillary permeability seen in implantation sites, and that its production by stromal cells may be the consequence of a paracrine action of epithelial prostaglandin, released under the effect of PAF and IL-1, derived from or produced by blastocysts in endometrial cells.

Hormonal anti-implantation agents: antiprogestins.

Oestrogens and progestogens separately or in combination are able to prevent implantation with high efficiency, thus acting as interceptive agents. Current interceptive medical regimens include high-dose oestrogens, the association of oestrogens with progestogens or progestogens alone. Compounds with antiprogesterone properties, such as RU 486 (mifepristone) or ZK 98734 (lilopristone), also exhibit a strong interceptive action which, as shown in animal models, is proportional to the dose and the day(s) of administration. Recent clinical studies show that RU 486 can be used successfully for postcoital interception. The regimen applied for this purpose consists of the intake of a single dose of 600 mg RU 486 within 72 h of a single act of unprotected intercourse. This treatment was found to be highly effective and to have a more favourably side-effect profile in comparison with the oestrogen-progestogen interceptive regimen. However, because of the induced irregularities of the cycle, the mifepristone regimen, as with the other hormonal methods, should not be used on a regular basis. Currently, all interceptive hormonal regimens are emergency methods. Their occasional use to prevent unwanted pregnancies may reduce the number of therapeutic abortions. However, the frequency and extent of their side-effects do not allow for a repeated postcoital use after every act of unprotected intercourse. Obviously, the development of an effective and safe 'morning after pill' requires further basic and clinical investigations.

Effects of cholic acid and 'Protectaid' formulations on human sperm motility and ultrastructure.

Cholic acid (sodium cholate) and the other active ingredients of F-5 gel preparations in use for the impregnation of a new vaginal sponge (Protectaid) with contraceptive and anti-sexually transmitted disease properties, were assessed for their effects on human sperm motility and ultrastructure. Cholic acid (CA) produced an inhibition of motility which was both dose- and time-dependent. A complete suppression of motility was obtained at 30 s by a CA concentration of 1.25%. Nonoxynol-9 (NX9) compared with benzalkonium chloride (BZC) showed no significant difference at the concentration required (0.025%) to give a total inhibition of sperm motility after exposure for 30 s. The addition of F-5A gel containing 0.5% of each one of the spermicide ingredients (CA, NX9 and BZC) produced the total suppression of sperm motility within 30 s at a dilution of 1/50. Another preparation, F-5B gel, containing the spermicide ingredients at different concentrations (1.25% CA, 0.125% NX9 and 0.05% BZC) produced this same effect with a 1/10 dilution. Exposure of semen to a CA concentration of 1.25% or to 1/10 dilutions of F-5A gel for 30 s led to profound changes of sperm ultrastructure studied by scanning (SEM) and transmission (TEM) electron microscopy. SEM and TEM findings indicate that CA acts as a spermicide through its 'natural detergent' properties, damaging the outer plasma membrane of sperm cells. Protectaid formulations affect sperm motility and viability in a similar way.

The zona reaction in human oocytes as seen with scanning electron microscopy.

Using scanning electron microscopy we found differences in the fine structure of the zona pellucida between unfertilized and fertilized human pronuclear stage oocytes in an in-vitro fertilization programme. In unfertilized oocytes, the zona pellucida appeared porous, comprising a large number of ring-shaped structures, called hoops, randomly superimposed in several layers. Superficial pores had a mean diameter of 4 microns, with the diameter decreasing in more inner lying pores. In fertilized oocytes, the zona pellucida was compact; the hoops appeared to melt and the pores to be obliterated by an amorphous material emerging from the inner zona. The micrographs provide ultrastructural evidence of the zona reaction in human oocytes and give insights into the morphological and mechanical aspects of the polyspermy-blocking mechanism in humans.

Compaction and surface polarity in the human embryo in vitro.

The surface morphology of the human ovum fertilized and cultured in vitro to the morula stage was studied by scanning electron microscopy with the specific aim of investigating embryo compaction and polarity. Unfertilized oocytes examined one day after attempted insemination (Day 0) were evenly and densely covered by long microvilli. The length and density of microvilli appeared to decrease in fertilized polypronuclear oocytes; a further decrease was observed in Day 2 and Day 3 embryos with 2-12 cells. No evidence of compaction or surface polarity was observed in any of these stages. On Day 4, compaction was evident in the majority of embryos with 10 or more cells, and the microvilli appeared dense again with a polarized distribution over the free surface of the compacted blastomeres. This study provides ultrastructural evidence that the human conceptus undergoes a relatively marked compaction at the morula stage during Day 4 postinsemination development in vitro.

Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles.

The formation of endometrial pinopodes detected by scanning electron microscopy may be a specific marker for uterine receptivity. Aiming to assess the effects of ovarian stimulation on pinopode formation, we examined sequential endometrial biopsies from 17 oocyte donors. Seven normally menstruating women served as controls. Up to four samples were taken from each woman at 24-72 h intervals between days 14 and 24, giving a total of 69 samples. The day of oocyte retrieval was designated day 14 in ovarian stimulation cycles and the day of luteinizing hormone surge was designated day 13 in natural cycles. Endometrial morphology and pinopode numbers were similar in both groups. Fully developed pinopodes appeared in only one sample per cycle, indicating their short life span. However, the cycle day these structures appeared varied up to 5 days between women and the distribution was as follows: day 18 (n = 2), day 19 (n = 7), day 20 (n = 4), day 21 (n = 3), day 22 (n = 1) in ovarian stimulation cycles, and day 20 (n = 2), day 21 (n = 2), day 22 (n = 3) in natural cycles. Furthermore, accelerated pinopode formation in ovarian stimulation cycles was positively correlated with day 13 progesterone. Our findings show that ovarian stimulation does not affect endometrial pinopode formation in terms of quantity and life span. The cycle days when pinopodes form are specific to the individual, being on average 1-2 days earlier in ovarian stimulation than in natural cycles. These changes in pinopode expression may reflect shifts in the window of receptivity, resulting in ovo-endometrial asynchrony and limiting implantation success in in-vitro fertilization.

Uterine pinopodes as markers of the 'nidation window' in cycling women receiving exogenous oestradiol and progesterone.

In 14 cycling women participating in an in-vitro fertilization (IVF) donation programme, we examined the timing of the 'nidation window' using as a stage-specific 'marker' the presence of fully developed pinopodes on the apical surface of the luminal uterine epithelium. Each woman received exogenous oestradiol from the second day of their cycle and progesterone starting on day 8 or day 15 of the oestrogenic treatment. The women underwent two biopsies during the same artificial cycle, on either days 6 and 9 or days 8 and 10 of the progesterone treatment. All patients to whom oestradiol was administered for 7 days prior to progesterone administration (n = 9), and two of the five treated with oestradiol for 14 days prior to the addition of progesterone, showed uterine pinopodes in either one or both biopsies. When present on a given day, pinopodes were at the same stage, developing, fully developed or regressing, showing that their total lifespan did not exceed 48 h. Fully developed pinopodes existed for 1 day only which may correspond to the short period of optimal endometrial receptivity observed in animal models. The timing of the presence of fully developed pinopodes varied from patient to patient, but these individual differences were not correlated with progesterone and oestradiol plasma concentrations. The brief duration of the nidation window and the observed individual variations in its timing suggest that the detection of uterine pinopodes could be a valuable tool for the prediction, on an individual basis, of the optimal date for successful egg replacement in IVF patients.