Pinopode Formation Research Articles

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 This article recapitulates published figures of endometrial ultrastructure and presents some unpublished data from ongoing studies.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.

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.

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.

Presence of uterine pinopodes at the embryo endometrial interface during human implantation in vitro

In order to study changes occurring on the surfaces of human endometrial epithelial cells in the presence of an implanted blastocyst, we used scanning electron microscopy for investigation of five endometrial biopsies and three human implantation sites obtained in vitro. All specimens showed areas with endometrial pinopodes, separated by cells displaying microvilli or cilia at the apical surface. Pinopode formation was more pronounced in endometrial biopsies than in cell cultures. All blastocysts adhered to pinopode presenting cells. Endometrial surface changes were not seen around the blastocysts. The results of this study demonstrate that cultured endometrial epithelial cells are capable of pinopode formation. Furthermore, endometrial epithelial pinopodes, generally considered as a marker of endometrial receptivity, seem to be directly involved in the adhesion of the blastocyst to the endometrial surface.

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.

Hormonal dependence of pinopode formation at the uterine luminal surface

The uterine luminal epithelium during the period of receptivity for nidation displays characteristic protrusions of the apical surface named pinopodes. The effects of oestradiol and progesterone, singly or in combination, on the formation and regression of pinopodes were investigated using scanning electron microscopy. The appearance of pinopodes was found to be strictly progesterone dependent. When given together with progesterone, before the development of pinopodes, high doses of oestradiol (plasma level approximately 300 pmol/l) inhibited pinopode formation; on the contrary, low doses of oestradiol (nidatory doses) did not interfere with the process until the 4th day of treatment. When oestradiol was given as a single injection, after pinopode formation, both doses were equivalent in inducing their regression 48-72 h later. It appears that the hormonal conditioning for pinopode formation and for the development of uterine receptivity for egg implantation is the same. These observations support the hypothesis that pinopodes could be an extremely useful tool to estimate uterine receptivity. The experiments we describe here, together with observations made a few years ago, in stimulated cycles in the human, suggest that implantation failure as a result of a hormonal imbalance during the time intervening between ovulation and nidation, may be a general phenomenon.