Ultrastructural markers of tissue endometrial receptivity in patients with recurrent implantation failure

Abstract

The scanning electron microscopy of the endometrial surface epithelium during the ‘implantation window’ was performed in 119 patients with uterine factor of infertility or recurrent miscarriage due to endometrial hypoplasia. Ultramorphological picture of the surface endometrial epithelium was characterized by aplasia and hypoplasia of pinopodes (67.39%), dense cell – cell contacts (69.53%), heteromorphy of secretory cells (15.22%) in combination with atypia of microenvironment cells (50%) in patients with infertility. The asynchronous development of pinopodes (46.67%) and the absence of intercellular contacts separation during the ‘implantation window’ (84.44%) was observed in patients with recurrent miscarriage. The revealed disturbance determines the mechanisms of the blastocyst adhesion violation and trophoblast invasion in the different stages of implantation in patients with uterine factor of infertility and recurrent miscarriage.

Keywords:

• Endometrial receptivity
• infertility
• implantation
• recurrent miscarriage
• pinopodes

Introduction

Impaired implantation of a human embryo due to a non-receptive endometrium is the most significant cause of reproductive failures of ART, occupying up to 70% in their structure during the transfer of good quality embryos [1,2]. Therefore, studies of the ultrastructural morphological characteristics of endometrial receptivity in the pathology of implantation are in the focus of scientific interest in recent years.

The purpose of the study was to estimate the ultrastructural picture of the tissue endometrial receptivity in patients with reproductive failure.

Material and research methods

The prospective observational comparative controlled study was performed on 119 women with uterine factor of infertility or recurrent miscarriage. The first group consisted of 92 women with uterine infertility factor due to endometrial hypoplasia. The second group consisted of 90 women with recurrent miscarriage due to endometrial hypoplasia. The control group consisted of 28 healthy fertile women who did not have a history of miscarriage, with a history of term delivery through the birth canal without deviating from the physiological course of pregnancy and the birth act that resulted in the birth of healthy children.

Inclusion criteria: reproductive age (18–45 years), uterine factor of infertility (for patients of the first group) or recurrent miscarriage (for patients of the second group) associated with endometrial hypoplasia, M-echo on day 19–22 of a cycle of 8 mm or less (for patients of the main groups) according to ultrasound data, ovulatory menstrual cycle.

Informed voluntary consent of the patient or her legal representative to participate in the study was obtained.

Exclusion criteria were: age less than 18 and more than 45 years old, oncological diseases, somatic pathology in which gestation of pregnancy is contraindicated, spouse karyotype abnormalities, other causes of infertility not related to endometrial hypoplasia.

Endometrial specimens taken by the pipelle-biopsy method were examined during the period of the proposed ‘implantation window’ (LH + 7).

After pretreatment of the endometrium and polymerization of the material, ultrathin sections were obtained on an UltraCutl 11 ultramicrotome, stained with lead citrate (Mi Honig, 1961) and examined in a JEM 100S electron microscope. For research in a scanning electron microscope, part of the material after dehydration was dried at the critical point of carbon dioxide sublimation and examined in a Hitachi-S350 raster electron microscope, Japan.

The statistical analysis was carried out using the application program Statistica 10 (Microsoft World). The results were processed using variation statistics and expressed in the form of M ± m. The confidence estimation of differences in average data and relative indices was carried out with the use of t-criterion (Student’s criterion). The significance point is factored as p < .05.

Results

The pattern of endometrial atrophy, the so-called ‘calm’ type of endometrium was observed more frequently in endometrial samples of patients with uterine infertility due to endometrial hypoplasia (Figure 1), there visualized a plots of pinopodes absents, the smooth apical surface of the glands epithelium, the gland in the state of ‘rest’, lack of secretory activity – in 67.39% of patients with uterine infertility, in the control group 14.28% (p = .001), in patients with miscarriage – 42.2%, which significantly exceeds the control values (p = .012) (Table 1).

Figure 1. “Calm” epithelium of the glands of the endometrium, a smooth apical surface and the absence of pinopodes, dense, well-marked cell-cell contacts. SEM of the surface epithelium of the endometrium.

Table 1. Ultrastructural morphological picture of the endometrium during the ‘window of implantation’ in the observation groups, (% ± m, χ², p).

Parameters	Infertility n = 46 %±m / abs.	Miscarriage n = 45 %±m / abs.	Control n = 28 %±m / abs.	χ^2 / р*
	1	2	3	χ^2 / р1–2	χ^2 / р1–3	χ^2 / р2–3
1. Pinopodes hypoplasia	52.17 ± 7.45%	37.78 ± 7.22%	14.28 ± 6.73%	1.904	10.620	4.648
	24	17	4	.168	.001	.031
2. Pinopodes absence	67.39 ± 6.98%	42.22 ± 7.36%	14.28 ± 6.73%	5.821	19.69	6.242
	31	19	4	.015	.0001	.012
3. Atypical ciliary cells	50.00 ± 7.45%	28.88 ± 6.75%	7.14 ± 4.95%	4.24	14.291	4.999
	23	13	2	.039	.0001	.025
4. Heteromorphic secretory cells	15.22 ± 5.45%	2.22 ± 0.2%	0%	7.148	4.706	0
	7	1	0	.006	.030	0
5. Acceleration and asynchronous pinopodia maturation	17.39 ± 5.69%	46.67 ± 4.73%	14.28 ± 6.73%	8.979	0.123	8.037
	8	21	4	.003	.725	.004
6. Intercellular contact disruption	69.56 ± 6.90%	84.44 ± 5.40%	7.14 ± 4.95%	2.837	27.307	41.639
	32	38	2	.092	.0001	.0001
7. Mature pinopodia	34.78 ± 7.02	55.56 ± 7.41	71.42 ± 8.69	3.965	9.356	1.839
	16	25	20	.046	.0022	.175
8. Maturing pinopodia	82.61 ± 5.9	66.67 ± 7.02	21.42 ± 7.89	3.061	27.26	14.13
	38	30	6	.080	.0001	.0001
9. Intensive secretion zone	23.91 ± 7.13	35.56 ± 7.14	92.85	1.806	13.191	7.221
	11	16	26	.423	.0001	.007

*Taking into account the Bonferroni amendment, when comparing the three observation groups, the differences are considered significant at a significance level of p < .017.

The lack of pinopodes maturation has always been accompanied by the presence of dense intercellular contacts in the surface epithelium of the endometrial glands, the absence of cell separation, which is a necessary condition for full implantation and invasion of the trophoblast. The microenvironment cells had the usual sizes and shape, the average density of their distribution was noted.

Pinopodes hypoplasia was characterized by rare non-uniform pinopodes fields on the background of zones of ‘quiet’ gland epithelium or irregular small pinopodes (Figure 2). At the same time, dense cell – cell contacts were observed. Microvilli on the apical surface of secretory cells are rare and shortened. The density of ciliary cells and their morphology were not changed. There were no zones of active secretion of endometrial glands and no secretion granules were visualized (Figure 2).

Figure 2.  Pinopodia hypoplasia: rare pinopodia on the background of “spontaneous” epithelium, polymorphic cells of the endometrial glands. Pronounced intercellular contacts. SAM. P – mature pinopodia, C – ciliary cells, E – erythrocytes.

Pinopodes hypoplasia during the ‘implantation window’ period was observed in 52.17% of women with uterine infertility and 37.78% of women with recurrent miscarriage, which was significantly higher than the level of control values (p1 = .001, p2 = .013) (Table 1).

Tight intercellular contacts were observed significantly more often in the main observation groups: 69.56% of cases in the group of women with uterine infertility and 84.44% of cases in the group of women with recurrent miscarriage. It was significantly more often than in the control group 7.14% (p1, 2<.001) (Table 1).

Acceleration and asynchronous nature of pinopodes maturation was more often observed in patients with recurrent miscarriage in 46.66% of cases, against 17.39% of cases in the group of women with infertility (p = .003), exceeding the control values of 14.3% more than three times (p = .004).

The morphological picture of this pathological process was associated with the maturation disorder of individual secretory cells of the endometrium, in which a combination of ‘calm’ epithelium gland fields with maturing pinopodes fields (uneven pinopodes maturation) or mature pinopodes were combined with fields of maturing pinopodes (maturation chronometric delay), or against the background of mature pinopodes fields, fields of intensive secretion of glands with many secretory granules (visualization of the epithelium transformation les endometrium). At the same time, there were normal signs of separation of cell-to-cell contacts. Ciliary cells were developed normally, their morphology, as a rule, was not changed (Figure 3).

Figure 3. Asynchrony of pinopodia ripening: “quiet” epithelium (SE) fields are combined with islands of mature (MP) and ripening (RP) pinopodia. SEM of the surface epithelium of the endometrium.

The presence of atypical ciliary cells was observed in 50.0% of women with infertility, which was significantly higher than in the samples of the endometrium in the control group – 7.14% (p = .0016) and exceeded the same indicator in women with miscarriage – 28.88%, however not reaching the level of confidence (Table 1). Ciliary cells had either an increased density in the field of secretory cells – hyperplasia of ciliary cells, or, on the contrary, low distribution density (hypoplasia of ciliary cells) and an atypical goblet shape according to our observations. This pathology is often combined with heteromorphic secretory cells, or asynchronous pinopodes maturation.

Heteromorphy of secretory cells was observed in 15.2% of women with infertility and only in one patient with a miscarriage (p = .006). In the control group this type of pathology was absent (Table 1). The pathological picture was characterized by different sizes, shape and structure of endometrial secretory cells. The sizes of cells differed two or more times. The cells, as a rule, had a different chronological picture of development. Such a structure of the glandular epithelium is often accompanied by the presence of atypical cells of the microenvironment, ciliary cells. It is possible that the correct ‘skeleton’ of the cell membrane and intercellular interactions, which form the configuration of endometrial secretory cells, depend on their structure and function.

The lag in pinopodes maturation, unevenness in maturation and development, asynchrony of these processes was observed in most women of the main observation groups – in patients with infertility – 82.61%, in women with miscarriage – 65.67%, which significantly exceeds the level of controls values – 21.4%; p1 = .0011, p2 = .0017.

Mature pinopodes occurred in 34.7% of women with infertility and in 55.56% of women with miscarriage, which was significantly lower than the control level – 71.42%; p1 = .002. Mature pinopodes were determined in endometrial specimens of women with uterine infertility and recurrent miscarriage due to endometrial hypoplasia, as a rule, only in combination with maturing pinopodes, had an uneven distribution on the surface of the endometrium, and most often were located in the mouths of the glands.

Discussion

The laminar epithelium of the endometrial glands is the first to ‘meet’ the blastocyst during the implantation period. Ultrastructural changes of its surface, studied using scanning electron microscopy, have opened a new level of endometrial receptivity – tissue. The decrease in the expression of receptors for estradiol and progesterone in the implantation endometrium coincides with a change in the ultrastructure of the apical surface of epithelial membranes with the formation of pinopodes [3]. It is on the pinopodes surface that the blastocyst adhesion occurs, potentiated by the interaction of LIF and LIFR, the expression of adhesion molecules (αvβ3), osteopontin and HB-EGF [4–10]. In our study, the ultrastructural morphological picture of the endometrium in patients with uterine infertility and recurrent miscarriage due to endometrial hypoplasia was characterized by a high frequency of pinopodia hypoplasia and aplasia during the ‘implantation window’, the presence of dense intercellular contacts in endometrial secretory cells, frequent asynchronous development and uneven maturation of pinopodes fields.

In patients with infertility in the ultrastructural picture of the endometrium, the fields of absence of pinopodes, their hypoplasia, heteromorphism of secretory cells in combination with atypia of microenvironment cells dominated. Mature pinopodes are rare and were distributed unevenly in the endometrial fields, mostly located in the mouths of the glands.

Microenvironment cells (ciliary cells) present on their surface a transmembrane epithelial mucin - MUC1, which is absent on the pinopodes surface. Its biological role is to repel the blastocyst and its correct orientation for invasion only in the pinopodia area [11].

In patients with miscarriage, the problem is associated with a chronometric lag in the maturation of pinopodes, their asynchronous development, as well as the lack of disconnection of cell – cell contacts during the ‘implantation window’. It has been demonstrated that one of the stages in the maturation of pinopodes is the disruption of the intercellular contacts of the surface epithelium, the ‘dissociation’ of cells [10]. The biological meaning of this phenomenon, is in our opinion, to ‘facilitate’ the invasion of the blastocyst into the endometrium. Mature pinopodes were visualized in half of the examined endometrial specimens in women with miscarriage, but their fields were also uneven.

Conclusion

The demonstrated disturbance could determine the mechanisms of violation of the blastocyst adhesion and the trophoblast invasion in different stages of implantation in patients with recurrent implantation failure.

Ethical approval

The research protocol was approved by the local ethics committee of the Federal Research Institute of Maternity and Child Care, Russian Ministry of Public Health, September 12, 2017, Paper No. 5.

Disclosure statement

The Study performed in the Federal Research Institute of Maternity and Child Care, Russian Ministry of Public Health as part of the federal state program for the research.

Authors report no conflicts of interest.