The role of mutations in the PI3K/AKT/mTOR signal pathway in decreasing ovarian reserve in reproductive patients with deep infiltrative endometriosis

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Endometriosis, according to various authors, is detected in 5-15% of women of reproductive age. One of the most common reasons for patients with endometriosis seeking medical help is infertility, which affects 25 to 50% of patients with this disease. The exact pathophysiology of infertility in endometriosis has not been established; a multifactorial effect of genetically and morphologically altered endometrioid tissue on the organs of the reproductive system is assumed [1-5].

Among the various factors that determine infertility or subfertility in women, the most important is the depletion of the pool of residual ovarian follicles [6].

Recent studies confirm a decrease in ovarian reserve in patients with genital endometriosis, regardless of the localization of endometrioid heterotopies [7-10].

The mechanism of a decrease in ovarian reserve in deep infiltrative endometriosis has not yet been established. Considering that endometriosis is a proliferative disease, the characteristic features of which are the ability to infiltrative growth, the absence of a pronounced capsule around the endometrioid focus, germination into neighboring organs, we can assume an increased antiapoptic activity of the ectopic endometrium in this disease. In recent years, the influence of apoptosis regulators on changes in the ovarian reserve in external genital endometriosis has been actively studied.

The role of the antiapoptic kinase signaling pathway PI3K / AKT / mTOR in the regulation of ovarian reserve is being studied. This is one of the universal intracellular signaling pathways characteristic of most human cells, which is responsible for avoiding apoptosis, cell growth and proliferation. This signaling pathway plays a significant role in the progression of endometriosis, activating proliferation and suppressing apoptosis of endometrioid cells [11].

Studies of mutations in the PIK3CA gene, which encodes the catalytic subunit of the phosphatidylinositol-3-kinase enzyme, have found wide application in the field of oncology, and the role of mutations in this gene in endometriosis has been little studied to date.

The aim of the study was to study the effect of deep infiltrative endometriosis on the ovarian reserve status in reproductive patients, as well as to assess the spectrum of mutations in the PIK3CA gene among patients with deep infiltrative endometriosis (DIE).

Patients and methods

A prospective cohort study was carried out that included 75 patients of reproductive age who were treated at the gynecological department of the Ural Research Institute of Maternity and Infancy Protection of the Ministry of Health of Russia in the period from 2019 to 2020. The main group consisted of 50 patients of reproductive age with deep infiltrative endometriosis, in 18 of whom deep infiltrative endometriosis was combined with ovarian endometriomas. Surgical treatment of patients of the main group was carried out by laparoscopic approach and consisted in maximum excision of endometrioid infiltrates within healthy pelvic tissues and removal of ovarian endometriomas by enucleation of the pseudocapsule of endometriomas with point precision bipolar coagulation of the vessels of the bed. In all patients included in the main group, the diagnosis was verified histologically.

The comparison group consisted of 25 patients of reproductive age who underwent laparoscopic metroplastics for inconsistency of the uterine scar from a cesarean section, with the exception of patients with endometriosis of an inconsistent uterine scar.

All patients signed written informed consent to participate in this study. The research protocol was previously discussed and agreed upon at the local ethics committee.

The analysis of the somatic and gynecological anamnesis, menstrual and reproductive functions, as well as the peculiarities of the clinical picture of the disease in patients of both groups was carried out.

In order to assess the state of the ovarian reserve in patients of both groups, the level of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and estradiol (E2) in the blood was determined by the enzyme-linked immunosorbent assay (ELISA) on days 2-5 of the menstrual cycle, as well as the number of antral follicles in the ovaries with transvaginal ultrasound (ultrasound) in the early follicular phase.

The search for PIK3CA gene mutations was carried out in tissue samples of ovarian endometriomas in patients with a combination of infiltrative endometriosis and endometrioid ovarian cysts (n = 18), as well as in biopsies of healthy ovarian tissue in all patients of the main group (n = 50) and the comparison group (n = 25) ). The most frequently described in the literature activating mutations of the PIK3CA gene were investigated: c.1624G> A (p.E542K), c.1625A> G (p.E542G), c.1625A> T (p.E542V), c.1633G> A ( p.E545K), c.1634A> G (p.E545G), c.1635G> T (p.E545D), c.1636C> G (p.Q546E), c.1636C> A (p.Q546K), c .1637A> C (p.Q546P), c.1637A> G (p.Q546R), c.3139C> T (p.H1047Y), c.3140A> T (p.H1047L), c.3140A> G (p .H1047R).

Genomic DNA was isolated from tissue samples using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics, Switzerland) using the MagNA Pure LC 2.0 Instrument (Roche Diagnostics, Switzerland). Subsequently, the stages of sample preparation were carried out before high-throughput parallel sequencing (next-generation sequencing - NGS) and NGS itself on the MiSeq sequencer (Illumina, USA) in accordance with standard protocols. The analysis of the quality of the obtained data was carried out using the DNA Sequence Assembler software (Heracle BioSoft S.R.L., Romania) and BWA-0.7.13 (Li H., Durbin R., Great Britain). Alignment to the human genome, as well as assembly of the sequence from reads, was performed using the BLAST program (National Library of Medicine, USA). The analysis of the prepared data was carried out using the MEGA v.7.0 software (Japan).

Statistical data processing was carried out using the application package, "Exel", "SPSS Statistics 22.0". For indicators characterizing qualitative characteristics, the absolute value and the relative value in percent were indicated; the chi-square test was used to test statistical hypotheses. Statistical hypotheses about the absence of intergroup differences were tested using the Student's t test.

Research results and their discussion

The average age of women in the groups was comparable and amounted to 33 ± 5.14 years in the main group and 33 ± 3.61 years in the comparison group (p> 0.05). When studying the anthropometric data of women in the observation groups, no significant differences were found, the average body mass index of the patients in the main group was 22.27 ± 3.73 kg / m2, in the comparison group - 23.46 ± 3.90 kg / m2, p> 0. 05.

Most of the patients in the main group (84%) had higher education, which is statistically significantly higher than the comparison group (52%), p = 0.004. Patients of the two groups were comparable in terms of the nature of concomitant somatic pathology. The smoking frequency among women was 8.0% in each group, p> 0.05. Age of menarche, duration of the menstrual cycle and duration of menstrual bleeding were comparable in the groups, p> 0.05. The painful nature of menstruation was observed in more than half of the patients of the main group (52%), which was significantly higher than in the patients of the comparison group (16%), p = 0.003. Violation of the menstrual cycle was noted by 16% of patients with endometriosis and 72% of patients with inconsistency of the uterine scar from a cesarean section (p <0.001), which is explained by the nature of the pathology.

The average age at the onset of sexual activity was also comparable in the groups, p> 0.05. There were no statistical differences in the frequency of use of combined oral contraceptives in the groups, p> 0.05.

24% of patients in the main group and 100% of patients in the comparison group (p <0.001) had at least one birth in the anamnesis, which is associated with the selection of women with a history of operative delivery to this group. There were no significant differences in the incidence of medical abortions, spontaneous miscarriages, non-developing and ectopic pregnancies in patients of both groups, p> 0.05.

The frequency of infertility was significantly higher in patients with endometriosis and amounted to 42% versus 16% in the comparison group, p = 0.025. At the same time, there is a predominance of primary infertility (76%) over secondary (24%) among patients with endometriosis-associated infertility.

When assessing the gynecological history, it was revealed that ectopia of the cervix was observed in 20% of patients in the main group, while in patients of the comparison group, ectopia of the cervix was not detected, p = 0.017. In 12% of patients, infiltrative endometriosis was combined with congenital malformations of the internal genital organs; in the patients of the comparison group, congenital malformations of the genital organs were not observed (p = 0.071). Uterine fibroids were more common in the main group (34%) than in the comparison group (20%), but the difference was not statistically significant (p> 0.05). Also, there were no significant differences in the incidence of other gynecological diseases in the two groups. 22% of patients in the main group had a history of surgical treatment of ovarian endometriomas, on average 4.5 ± 1.07 years ago. Surgical excision of endometriosis foci in anamnesis underwent 12% of patients in the main group on average 2.7 ± 1.43 years ago. When assessing the history of other gynecological operations, there were no significant differences in the two groups.

Patients with deep infiltrative endometriosis were more likely to suffer from chronic pelvic pain (82%) than patients in the comparison group (16%), p <0.001. Dyspareunia was also observed more often in the main group (30%) than in the comparison group (8%), p = 0.032. Dyschezia and dysuria occurred in 4% of patients in the main group, in the patients in the comparison group these symptoms were not observed, p> 0.05.

When assessing the state of the ovarian reserve in the patients of the two groups, it was found that the AMH level was lower in the patients with the infiltrative form of external genital endometriosis than in the patients of the comparison group by an average of 1.0 ng / ml (2.6 ± 2.2 ng / ml in the main group, 3.6 ± 3.5 ng / ml in the comparison group), however, the difference did not reach statistical significance, p> 0.05. In patients of early reproductive age (up to 35 years), the difference in the average AMH level between the two groups was more pronounced and amounted to 1.5 ng / ml (2.8 ± 2.1 ng / ml in the main group, 4.3 ± 4, 0 ng / ml in the comparison group), however, it also did not reach statistical significance, p> 0.05. It should be noted that there was no statistically significant difference in the AMH level in patients with an infiltrative form of external genital endometriosis combined with ovarian endometriomas (2.4 ± 1.9 ng / ml) and not combined with ovarian damage (2.7 ± 2 , 4 ng / ml), p> 0.05.

The mean FSH level did not significantly differ in the patients of the two groups, amounting to 6.8 ± 4.2 mIU / ml in the main group and 7.6 ± 10.3 mIU / ml in the comparison group, p> 0.05. With the exclusion of patients of late reproductive age (over 35 years), there was a slight predominance of the average FSH level in the main group (6.8 ± 4.3 mIU / ml) than in the comparison group (4.9 ± 3.4 mIU / ml) however, the difference was not statistically significant. Also, there were no statistically significant differences in the average level of estradiol in the blood between the main group (148.8 ± 128.8 pg / ml) and the comparison group (183.4 ± 125.4 pg / ml), p> 0.05, although its lower level in the main group attracts attention.

The number of antral follicles according to transvaginal ultrasound was significantly lower in the main group (8.5 ± 4.5) than in the comparison group (12.2 ± 4.1), p = 0.001. It should be noted that this difference was statistically significant both for patients with ovarian endometriomas (6.0 ± 4.2, p <0.001) and for patients without ovarian endometriosis (9.8 ± 4.2, p = 0.04). Data reflecting the state of the ovarian reserve in patients of two groups are presented in Table 1.

  Table 1 - The status of the ovarian reserve in patients of observed groups

Indicator	Main group (n=50)	Comparison group (n=25)	р
Anti-Müllerian hormone, ng / ml	2,6±2,2	3,6±3,5	0,25
Follicle-stimulating hormone, mIU / ml	6,8±4,2	7,6±10,3	0,72
Estradiol, pg / ml	148,8±128,8	183,4±125,4	0,28
Number of antral follicles	8,5±4,5	12,2±4,1	0,001*

Note. Data are presented as mean and standard deviation (M ± s). * -data are statistically significant

 

The data obtained indicate a decrease in the ovarian reserve in patients of reproductive age with an infiltrative form of external genital endometriosis, regardless of the presence of endometrioid lesions of the ovaries. The mechanism of a decrease in ovarian reserve in endometriosis, especially in the absence of visible damage to ovarian tissue, is not fully understood. In recent years, the role of regulators of apoptosis in reducing the ovarian reserve in endometriosis has been actively studied.

Recently developed by Kawamura K. et al. the method of in vitro activation of primary follicles in patients with primary ovarian failure drew attention to the role of the antiapoptic kinase signaling pathway PI3K / AKT / mTOR in the regulation of ovarian reserve [11].

The PI3K signaling pathway plays an important role in the activation of primordial follicles, which is highlighted in studies on transgenic animals [12].

In their study, Takeuchi A. et al. (Japan, 2019) demonstrated that in endometriosis, the proportion of primordial follicles is reduced, and the proportion of primary, secondary, antral and growing follicles is increased compared to the control according to histological examination of ovarian tissue. It was shown that in endometriosis, the PI3K / AKT / mTOR signaling pathway is activated according to immunohistochemical studies. Administration of AS101, an inhibitor of the PI3K / AKT / mTOR signaling pathway, restored the proportion of primordial follicles in the mouse model of endometriosis to the level of the control group. These results indicate a significant role of the PI3K / AKT / mTOR signaling pathway activated in endometriosis in the premature depletion of the pool of primordial follicles and a decrease in the ovarian reserve in patients with endometriosis [13].

To date, the role of PIK3CA gene mutations in the pathogenesis of breast, endometrial, cervical, colon, lung cancer, and ovarian cancer has been described [14].

Atypical ovarian endometriosis degenerates into endometrioid or clear cell carcinoma in 15–32% of cases. Mutations in exons 10 (9) and 21 (20) of the PIK3CA gene are detected in 20% of endometrioid and clear cell ovarian carcinomas and are an unfavorable prognostic factor [15].

In recent years, there have been publications of studies on the role of mutations of oncogenes in the pathogenesis of non-oncological diseases, including endometriosis [1617].

Michael S Anglesio et al., 2019, performed exome sequencing for the presence of somatic mutations in patients with infiltrative endometriosis, which has the lowest risk of malignancy. Exome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients had known mutations of the PIK3CA, ARID1A, KRAS, or PPP2R1A oncogenes [18].

V Lac et al., 2019, in a multicenter retrospective study determined the presence of mutations of oncogenes involved in the signaling pathways MAPK / RAS and PI3K / AKT / mTOR, in the foci of postoperative endometriosis of the anterior abdominal wall (n = 40) and foci of deep infiltrative endometriosis (n = 36) using new generation DNA sequencing, drop PCR and immunohistochemical analysis. Overall, somatic mutations of oncogenes were detected in 11 out of 40 (27.5%) cases of postoperative endometriosis and in 13 out of 36 (36.1%) cases of deep infiltrative endometriosis, including point mutations in KRAS, ERBB2, PIK3CA, and CTNNB1. There were no statistically significant differences in the frequency of detected somatic mutations between postoperative and deep infiltrative endometriosis (P> 0.05) [19].

We isolated and analyzed DNA regions of exon 10 (9) (1540-1664 nucleotide sequences, 514-554 amino acids) and 21 (20) exons (3030-3185 nucleotide sequences, 1011-1061 amino acids) of the PIK3CA gene in forward and reverse sequences. The average number of reads along the forward and reverse sequences of DNA sections was 1629.4 ± 340.5, which corresponds to a good quality of readings (Figures 1, 2).

Figure 1. Assessment of the quality of reading the forward (a) and reverse (b) DNA sequences of exon 10 (1540-1664 bp) and part of exon 21 (3030-3185 bp) of the PIK3CA gene (x - average reading quality, y - number of reads; performed using NextGen Sequence Workbench)

Figure 2. Basic content of the forward (a) and reverse (b) DNA sequences of exon 10 (1540-1664 bp) and part of exon 21 (3030-3185 bp) of the PIK3CA gene (performed using the NextGen Sequence Workbench program)

Our study did not reveal PIK3CA gene mutations in any of the tissue samples of ovarian endometriomas in patients with a combination of infiltrative endometriosis and endometrioid ovarian cysts (n = 18), as well as in none of the biopsies of healthy ovarian tissue in patients of the main group (n = 50) and the comparison group (n = 25) using the next generation DNA sequencing method.

In an earlier study, Yang Zou et al. (China, 2018) identified a total of four somatic mutations in three out of 101 ovarian endometrioma samples (4%, 4/101), including KRAS p.G12V, PPP2R1A p.S256F, and two ARID1A nonsense mutations (p.Q403 and p .G1926); while in the remaining 7 genes (BRAF, NRAS, HRAS, ERK1, ERK2, PTEN, and PIK3CA), no mutations were detected. In addition, no mutations were detected in any of the 10 genes, either in the eutopic endometrium of patients without endometriosis (n = 85), or in the healthy ovarian tissue of patients with endometriosis (n = 62) [20].

Perhaps the small sample size did not allow us to identify mutations in the PIK3CA gene in the examined patients, and large-scale population studies are needed to identify mutations in this gene in endometriosis. Also, activation of the PI3K / AKT / mTOR signaling pathway in endometriosis can be caused by mutations of other genes encoding proteins regulating the PI3K / AKT / mTOR signaling pathway, such as tyrosine kinase c-kit, protein kinase B (AKT), transcription factor FOXO3, phosphatase and homolog tensin (PTEN), mammalian target of rapamycin (mTOR), which requires further research.

Conclusion

Thus, the presence of deep infiltrative endometriosis is associated with a decrease in ovarian reserve in patients of reproductive age, regardless of the presence of endometrioid ovarian lesions.

Our study did not reveal any known activating mutations in the PIK3CA gene among patients with infiltrative external genital endometriosis.

Population studies are needed to identify mutations of this gene in endometriosis, as well as to study mutations of other genes encoding proteins regulating the antiapoptic signaling pathway PI3K / AKT / mTOR, to identify the mechanism of depletion of the ovarian reserve in infiltrative form of external genital endometriosis.

About the authors

Oxana A. Melkozerova

Urals Scientific Research Institute for Maternal and Child Care

Author for correspondence.
Email: abolmed1@mail.ru
ORCID iD: 0000-0002-4090-0578
Scopus Author ID: 56358107200
ResearcherId: AAA-6978-2020
http://niiomm.ru/administratsiya-nii-omm/554-melkozerova-oksana-aleksandrovna

D. Sci. (Med.)

Russian Federation, 620028, Ekaterinburg, Repin street, 1

Ekaterina O. Okulova

Urals Scientific Research Institute for Maternal and Child Care

Email: cat93_07@mail.ru
ORCID iD: 0000-0002-3035-2862

Graduate Student

Russian Federation, 620028, Ekaterinburg, Repin street, 1

Anna A. Mikhelson

Urals Scientific Research Institute for Maternal and Child Care

Email: ann_lukach@list.ru
ORCID iD: 0000-0003-1709-6187
Scopus Author ID: 57211026709
ResearcherId: AAB-9373-2020

D. Sci. (Med.)

Russian Federation, 620028, Ekaterinburg, Repin street, 1

Tatyana B. Tretyakova

Urals Scientific Research Institute for Maternal and Child Care

Email: tbtretyakova@yandex.ru
ORCID iD: 0000-0002-5715-7514
SPIN-code: 8626-2640
Scopus Author ID: 57191620822

Cand. Sci. (Med.)

Russian Federation, 620028, Ekaterinburg, Repin street, 1

References

Supplementary files