Markery ryzyka zaburzeń funkcji rozrodczych u kobiet z hiperandrogenizmem

Ludmyla M. Semeniuk1, Volodymyr K. Likhachov2, Tetyana Y. Yuzvenko1, Ludmyla М. Dobrovolska 2 , Oleg G. Makarov2

1Ukrainian Scientific and Practical Center of endocrine surgery, endocrine transplantation of organs and tissues
of the Ministry of Health of Ukraine, KYIV, Ukraine

2Ukrainian Medical Stomatological Academy, Poltava, Ukraine

ABSTRACT

Introduction: The frequency of the pathological course of pregnancy with hyperandrogenism ranges from 21 to 48%. With the predominance of androgens against the background of lowering the normal products of steroidogenesis, a pro-inflammatory immune imbalance is formed, which causes a premature termination of the development of pregnancy. Many studies indicate that hyperandrogenism is a factor in the development of obstetric pathology, including placental dysfunction. The severity of obstetric complications of pregnancy in women with hyperandrogenism depends on the duration of the disease and the quality of preparation for pregnancy. Establishing early markers of endocrine hyperandrogenic imbalance in the body of a future mother and conducting timely therapy is the key to the successful completion of pregnancy.

The aim: the establishment of hormonalimmunological changes and morphological and receptive structure peculiarities of the endometrium correlations in women with PCOS at the pregnancy planning stage.

Materials and methods: 139 women with PCOS were screened at the stage of pregnancy planning. Diagnosis of PCI was conducted in accordance with the International Recommendations of Endocrine Society 2013 and the Rotterdam criteria of 2003. Endometrial aspiration was studied for the study of T and B lymphocyte populations, cytokine levels and endometrial receptors. Hormonal parameters were determined using the radioimmunological method using Immunotech (Czech Republic) and Siena Ire Sorin (France) kits. Normal values of the studied hormones in accordance with the recommendations of the manufacturer.

In order to study the populations of T and B lymphocytes and endometrial receptors the aspirate, obtained by endometrial biopsies, was used taken on the 5-7 and 21-24 days of the menstrual cycle. The cytokines levels (INF-y, TNF-a, Il-4, Il-6, Il-8, Il-10) were estimated in the endometrium with the help of the test system “Protein contour” on immunoenzymatic analyzer “Multyskan EX” (Germany). The assessment of estrogens receptors at stroma and glands and progesterone receptors at stroma and glands was performed by H –Score system, lymphocytes count was carried out by the light microscope at 400 x 3 fields-of-view expansion.

Statistical material analysis was carried out on the personal computer using the standard software package with applied statistical analysis (Excel 2003 and Statistica v. 6.0). The assessment of variables connection was carried out by Pirson’s coefficient correlations. The statistical significance of differences was performed by the use of nonparametric Mann –Whitney criteria, χ2 and Fisher criterium. The significance threshold of statistical value was considered p = 0.05.

Results: In women with hyperandrogenism, there was a significant increase (p <0.05) in the level of DHEA-S, a decrease in the level of E2 and progesterone in the early follicular phase compared with the control group and an increase in body weight by 37.5%.

At histological examination of the endometrium on the 5-7th day of the menstrual cycle there was a lag proliferative changes of the endometrium, and in one third of women hyperplastic process against the background of reduced proliferative activity. At GA there was a marked violation of expression of receptors as estrogens and progesterone stroma in all stages of the menstrual cycle. In women with hyperandrogenism, changes in the immunological profile of endometriosis with the prevalence of Th-1 orientation of immunity have been detected.

Conclusions: In women with hyperandrogenism there is a close correlation between the biochemical form of the domination of androgens, ovulatory dysfunction, immunohistochemical and morphological structure of the endometrium. Revealed features of pro-inflammatory direction of cytokine immunity.

Wiad Lek 2018, 71, 8, -1553

Introduction

Hormonal imbalance causes about 10 -15% of all pregnancy habitual loss cases [1]. The frequency of the pathological pregnancy progress with hyperandrogenism ranges from 21 to 48% [2]. If the androgens dominance occurs women experience proinflammatory immune imbalance as the result of normal steroidogenesis products decrease, causing premature gestation termination [2].

Many studies have indicated that hyperandrogenism is the main factor of obstetric pathology, including placental dysfunction [3]. The severity of pregnancy obstetric complications in women with hyperandrogenism depends on disease remoteness and quality of preconception period that woman had received before. [4].

The aim

Аim: the establishment of hormonal-immunological changes and morphological and receptive structure peculiarities of the endometrium correlations in women with PCOS at the pregnancy planning stage.

Materials and methods

The study group included 139 women with PCOS, experiencing the menstrual cycle violation and pregnancy planning. The control group involved 30 healthy women with normal menstrual cycle, and hormonal profile characteristics within normal limits. The international Endocrine society guidance 2013 and the Rotterdam criteria 2013 were taken as the biochemical criteria of PCOS diagnostics. The total (Tt) and free testoseronе(Тf), androstenedione(An), 17-hydroxyprogesterone(17-OHP), dehydroepiandrosterone sulphаte (DHEA-S), dehydrotestosteronе (DHT) ELISA levels were estimated[5]. The levels of estradiol and progesterone were estimated on the 5-7 and 21-24 days of the menstrual cycle. The investigated women were divided into groups according to the hyperandrogenism source: Group I – 28 women with elevated levels of testosterone, II group – 79 women with elevated levels of androgens, III group – 32 women with elevated dehydrotestosteronе.

In order to study the populations of T and B lymphocytes and endometrial receptors the aspirate, obtained by endometrial biopsies, was used taken on the 5-7 and 21-24 days of the menstrual cycle. The cytokines levels (INF-y, TNF-a, Il-4, Il-6, Il-8, Il-10) were estimated in the endometrium with the help of the test system «Protein contour» on immunoenzymatic analyzer «Multyskan EX» (Germany). The assessment of estrogens receptors at stroma and glands and progesterone receptors at stroma and glands was performed by H –Score system, lymphocytes count was carried out by the light microscope at 400 x 3 fields-of-view expansion.

Statistical material analysis was carried out on the personal computer using the standard software package with applied statistical analysis (Excel 2003 and Statistica v. 6.0). The assessment of variables connection was carried out by Pirson’s coefficient correlations. The statistical significance of differences was performed by the use of nonparametric Mann –Whitney criteria, χ2 and Fisher criterium. The significance threshold of statistical value was considered p = 0.05.

Results

Among the women of groups I and II were not noted significant differences in body mass index relatively to the women of the control group (21,4 ± 0,97 and 21,9 ± 1,2 to 21,06 ± 1,02 in control, p> 0.05 ). The following has demonstrated a significant (p <0.05) prevalence levels of DHEA-S (6,78 ± 0,65 – in group I and 6,98 ± 0,53 mmol / l – in the group II) and 17 -OHP (10,00 ± 0,25 nmol / l and 11,38 ± 0,13 nmol / l, respectively) compared with the control group (DHEA-S 3,34 ± 0,42 mmol / l 17-OHP 2, 94 ± 0,39 nmol / l). The content of ovarian and adrenal androgens in blood was within normal limits in the 3d group of women. The obesity (BMI 28,2 ±1,12) was determined among the 12 women of the following group.

The E2 reduction on the 5-7 days of menstrual cycle (11,8 ± 1,12 to 56,7 ± 1,98 nmol / L in the control group; p <0,05) was characteristic to all surveyed women with hyperandrogenism. The progesterone levels were reduced in all women with anovulatory menstrual cycles and it was reduced in the early proliferative (0,89 ± 0,15 nmol / L versus index control group 1,19 ± 0,31 nmol / L). The androgens levels were elevated in women with anovulation relatively to the control group: Tt. (7,17 ± 0,26 to 2,1 ± 0,78 nmol / L, p <0,005), Tf. (15,1 ± 1,32 to 17,2 ± 1,87 pmol / l, p <0,05), DHEA-S (22,7 ± 1,32 to 3,34 ± 0,42 nmol / L, p <0,005), An (5,5 ± 1,02 to 4,78 ± 0,92nmol / l, p = 0,01). During the luteal phase deficiency the levels of 17-OHP on the 21-24 days (9,7 ± 0,67 to 3,0 ± 0,21 nmol / L control group), DHT (403,5 ± 21,34 to 418,29 ± 30,59 p = 0,01) were increased in relation to the 1-st phase of the menstrual cycle, whereas with anovulation these figures virtually do not change (p > 0.05).

The DHT levels indicated the direct correlative affinity between the levels of DHT and Tf (e = 0.061, p <0.05) among the women of group III.

The histological endometrium examination on the 5-7 days of the menstrual cycle of the women from the I and II groups with the anovulation indicated the lack of proliferative changes in the endometrium. The hyperplastic process as the result of reduced proliferative activity was marked in 29.1% of women in group II. The study of the endometrium structure on the 21 – 24 days of menstrual cycle in women with LPD indicated the defective secretory transformation of the endometrium.

The receptor component of the endometrium immunohistochemical study in women with hyperandrogenism demonstrated receptor violation of estrogens and progesterone stroma as on the 5-7 and so on the 21-24 days of the menstrual cycle. The direct correlation between the levels of E2 in serum and progesterone receptor expression both in glands and stroma on the 5-7 days of menstrual cycle (ґ = 0,57, p <0.05) was indicated in women group I. The correlation rates reached ґ = 0,66 for glands and stroma to ґ =0.75, p <0,01 in women of group II. The inverse correlation between the low levels of progesterone and progesterone receptor expression increased for stroma and glands (ґ-0,66, p <0,05; and ґ-0,53, p <0,05 in accordance) was typical for the women of the following group.

The inverse correlation between the levels of total T, An, and 17 OHP in blood and progesterone receptors expression glands (PRg) (ґ-0,75, p = 0,01, and ґ-0,85; p <0,01 and ґ-0,89; p <0,05, respectively.), between levels. Tf and the expression of PRs (ґ-0,78; p <0,05), between levels 17 –OHP and expression REg (ґ-0,97; p <0,001) was marked in the women of the following group. The direct correlation between high levels of expression of RPs and Tf. (ґ = 0,84, p <0,01) was indicated among the women of group I.

The following changes were revealed while the study of the endometrium immunological profile in women with hyperandrogenism: a violation of phenotypic endometrial NK – cells with increased CD 56 +NK cells on the 5-7 days of the menstrual cycle and decline of CD 56+ NK cells on the 21-24 days of the menstrual cycle. The combination of CD16 + NK- cells on the 5-7 days of the menstrual cycle prevailed in women’s group I, which had a direct correlation with the level Tt (ґ = 0,81, p <0,01) and estradiol (E2 levels (ґ = 0,68, p <0,05) and an inverse correlation with reduced levels of progesterone in the blood. (ґ-0,60; p <0.05). The direct correlation between the increased number of CD 16+ NK – cells in the endometrium on the 5-7 days of the menstrual cycle and DHA-S levels in the blood (ґ = 0,62, p <0.05) was identified among the women group II. The positive direct correlation between increased levels of DHT in the blood and the number of CD 16+ NK – cells in the endometrium (ґ = 0,73, p <0,05) occurred on the 5-7 days of the menstrual cycle among the women of group III. The inverse correlation between the number of CD 16+ NK – blood cells and CD 16+ NK – cells in the endometrium (ґ-0,68; p <0,001) was marked in the women of group I.

The prevalence of proinflammatory Th-1 aimed immunity is marked among the women with hyperandrogenism. The levels of interferon -γ (INF- γ), and TNF-α and INF-γ among the women of group III exceeded the rates of the control group (p <0,05). The direct correlation between the levels of serum TNF-α and Tt., and An (ґ = 0,62, p <0,05, and ґ = 0,88, p <0,001 respectively) was marked. The decrease in the number of B cells and induced production of IL-2 and IL-4 was characteristic to the women with hyperandrogenism. The inverse correlation between the absolute number of B-lymphocytes in the blood and serum levels of DHT (ґ-0,58; p <0,05) was defined for the women from group III. The direct correlation between induced production of IL-4 and blood levels of B cells (ґ = 0,95, p <0,001) was defined for the women from group I.

Discussion

The revealed changes in hyperandrogenic dysfunction contribute to the formation of inability and endometrial receptors to gestational transformation and to focus proinflammatory immune responses on the endometrium. The work of other researchers regarding the endometrium of women with reduced fertility confirmed an increase in the level of proinflammatory cytokines[6].

The authors note the systemic nature of changes in the processes of regeneration of cellular and intracellular rearrangement of endometrium in women with endocrinopathies, which corresponds to the formation of regenerative-plastic deficiency syndrome[7].

In our study, as well as in the study of other authors, we came to the conclusion that the restoration of fertility in women with hyperandrogenism is associated with a violation of immune-endocrine-tissue relationships in the genital tract of women[8].

Therefore, today, the strategy of treatment of habitual miscarriage is induction of regenerative reactions in the epithelial and endothelial cell populations of the endometrium, which contributes to enhancing biosynthetic reactions, activating receptor sensitivity and positive structural and functional transformations of the endometrium[9].

The study allowed us to identify markers of future reproductive losses in patients with hyperandrogenism, which demonstrate a violation of the endometrial morphofunctional structure and demonstrate its inability to gestational adjustment.

The close correlations between the hormone-immune imbalance form in women with the hyperandrogenism were identified which indicates the feasibility of immunomodulators usage at the pregnancy planning stage.

Conclusions

1. There is a close correlation between the biochemical form of the domination of androgens, ovulatory dysfunction, immunohistochemical and morphological structure of the endometrium.

2. In women with hyperandrogenism revealed features of pro-inflammatory direction of cytokine immunity, which is the basis for the use of immunomodulators in the preparation for pregnancy.

References

1. Dobrokhotova Yu.E., Ragimova Z.Z., Ilina I.Yu., Ibragimova D.M. Hyperandrogenism and female reproductive health Moscow: GEOTAR-Media; 2015. 144р. (in Russian).

2. Goodman, N.F., Cobin, R.H., Futterweit, W., et al.“American Association of Clinical Endocrinologists, American College of Endocrinology, and Androgen Excess and PCOS Society Disease State Clinical Review: Guide to the Best Practices in the Evaluation and Treatment of Polycystic Ovary Syndrome. Part 2.Endocr Pract 21.12 (2015): 1415–26.

3. Brito JP, Domecq JP, Murad MH, Guyatt GH, Montori VM. The Endocrine Society guidelines: when the confidence cart goes before the evidence horse. J Clin Endocrinol Metab. 2013 Aug;98(8):3246-52. doi: 10.1210/jc.2013-1814. Epub 2013 Jun 19.

4. Azziz R. Controversy in clinical endocrinology:diagnosis of polycystic ovarian syndrome: the Rotterdam criteria are premature. J. Clin. Endocrinol. Metab. 2006; 91 (3): 781–785

5. Faculty of Sexual & Reproductive Healthcare of the RCOG.UK Medical Eligibility Criteria for Contraceptive Use (UKMEC 2016). Available from: [http://www.fsrh.org/pdfs/UKMEC2016.pdf], last accessed Aug 26, 2016.

6. L.V. Chernobai, O.M. Tyshenco, V.V.Lazurenko, I.Yu.Muryzina, O.V.Yurcova The role of citokin profile isorders in fertility reproduction Internationalmedicaljornal/ 2016; 2. 2016 38 -43. (In Ukrainian).

7. Fluker M., Fisher S. Anovulation and ovulatory dysfunction. In: Falcone T, Hurd WW. Clinical reproductive medicine and surgery. New York. 2007; 277-86.

8. Semeniuk L., Laryna O. Early prognostic markers for future reproductive losses at PCOS. In the 24th World congress of contraversies in Obstetrics, Ginecology &Fertylity (COGI) Amsterdam., The Netherlands, November 10-13/, 216 Abstract Book. 2016. 181

9. Bogavac M., .Jakovijevic А.,Gruji. Z. Biochemical markers – predictors or causes of complications in pregnancy. In the 18th World congress of contraversies in Obstetrics, Ginecology &Fertylity (COGI) Vena. Austria, October 24 – 27., 2013 Abstract Book. 2013. 91 – 92

The paper has been written within the research scientific work made at the Department of Obstetrics and Gynecology II of HSEEU “UMSA” within 2017-2022, entitled “The role of chronic uterine infection and lower genital tracts in the origination of obstetric and gynecological pathology” (State registration No. 0117U005276).

Authors’ contributions:

According to the order of the Authorship.

Conflict of interest:

The Authors declare no conflict of interest.

CORRESPONDING AUTHOR

Volodymyr K. Likhachov

42 Stritenska Str., app. 19, Poltava 36011, Ukraine

tel: +380952212112

e-mail: vladimir.lihachov@gmail.com

Received: 29.07.2018

Accepted: 14.11.2018