THE LEVEL OF ANTIOVARIAN ANTIBODIES AS A INDEX OF INFERTILITY

Introduction. In the structure of infertile marriage the main place has still been kept by a tubal factor. The frequency of this infertility factor reaches 18¬73%. Target of the study. To determine the level of antiovarian antibodies in the blood of women with tubal infertility. Materials and methods. A clinical-laboratory examination has been made of 109 women which were divided into 2 groups:1 group (main) - 58 women with infertility of tubal origin; 2 group (control) - 51 women. The antiovarian antibodies were determined by means of immunofermental set made by the firm Bioserv Ovari-Antibodi ELISA for determination in the blood serum of autoantibodies directed against ovarian antigenes. Results. 30 women have been examined and divided in two groups in our study. The first group included 20 women with infertility of tubal origin. The second group consisted of 10 healthy women with a preserved reproductive function. The average age of women in the main group didn’t differ from the women’s age of the control group. Significant majority in the number of women with chronic salpingoophoritis in the main group (6 times more) testified to the possibility of autoimmune salpingoophoritis initiation in the group under study. The level of antiovarian antibodies was determined equal to 7,1±0,9 and in the main group - 4,1±0,5, which is 1,7 times less than in the control group. Conclusions. 1. The level of antibodies in the main group of women under study and rise of the level have been determined in patients with a secondary infertility who didn’t impregnate for more than 5 years and underwent the following surgery; tubectomy, cystectomy, adnexectomy. 2.On the contrary, the decreased level of antiovarian antibodies has been discovered in patients with a primary infertility who did not impregnate for 5 years as well as in women with uterine pathology and myoma.Кафедра акушерства та гінекологі

TREATMENT OF AUTOIMUNNE OVARIAN DAMAGE

Introduction. To investigate levels of antiovarian autoantibodies in girls and young women with disturbances of menstrual cycle before and during treatment with hormonal therapy. To explain morphological changes in ovarian structure in these patients. Menopause usually occurs approximately at the age of 50. Premature ovarian failure (POF) is a disorder defined as a pathologic termination of menstrual cycle after puberty and before the age of 40. Frequency of this disorder is approximately 1%. Hormonal levels show hypergonadotrophic hypoestrism (FSH more than 40 lU/l). The onset of the disease may be very slow. Menarche and regular menstrual cycles may be followed by menstrual cycle disorders - oligomenorrhoea or secondary amenorrhoea. Sterility or infertility at the reproductive age could be manifestations of the early stage of the disease. One of the possible causes of premature ovarian failure could be an autoimmune process beginning at any time during the reproductive period. Autoimmune damage of the ovarian hormonal production places this disease between the autoimmune endocrinopathies, characterized by direct destruction of the target cells, such as thyroiditis, insulin dependent diabetes and Addison’s disease. Animal models proved that the manifestation of the disease is preceded by complex process that is influenced by genetic, immunologic and environmental factors. Material and methods. Studied group included 39 patients. 18 patients were treated for primary amenorrhoea, 21 for menstrual cycle disorders. Patients included in the study were repeatedly examined at the beginning of the study and after six months during which they were treated by estrogen and gestagen. In all patients we have tested FSH, LH and FSH/LH ratio, presence of antiovarian antibodies. Results were compared with those obtained in control women. 21 antiovarian antibodies positive patients were indicated for laparoscopic biopsy. Bioptic sample was examined using light and electron microscopy. Results. Our treatment with hormonal therapy lead to the reduction of ovarian antigens. In 85% of the cases marked decrease of antiovarian autoantibodies levels was observed, while in 28% of the cases the levels were undetectable. From morphological changes of the bioptic sample enhanced atresia of follicules at different developmental stages was frequently observed. It evoked marked reduction of follicular apparatus up to its complete disappearing. Conclusion. The results of our study and mapping of the antiovarian antibodies positivity support our hypothesis that the antiovarian antibodies positivity corresponds with the clinical symptoms. Appropriate treatment with hormonal replacement therapy minimizes ovarian destruction, preserves ovarian hormonal functions and saves healthy ovarian tissue.Кафедра акушерства та гінекологі

PECULIARITIES OF PREGNANCY, CHILDBIRTH AND THE POSTPARTUM PERIOD IN WOMEN WITH ENDOMETRIOSIS

Endometriosis - a widespread disease in women of reproductive age and perimenopausal. Endometriosis is a chronic progressive disease and recurrent disease, which affects 12 to 60% of women of reproductive age. In women with different forms of endometriosis pregnancy occurs according to the latest research with significant abnormalities. According to other authors, 30 - 40% of patients with endometriosis suffer from infertility. Objective. To evaluate the peculiarities of pregnancy, childbirth and the postpartum period in women with endometriosis. Material and methods. In Chernivtsi city maternity hospital №1 were examined 60 pregnant women of reproductive age from 21 to 42 years (average age was 31,5 ± 3,26 years), the main group included 30 women with clinical signs of endometriosis and in control - 30 women without gynecological and somatic disorders. The study of pregnancy in the examined groups showed that in the study group threatened miscarriage occurred 5,9 times more frequently than among patients in the control group. In assessing obstetric complications was found that 76,7% of women with endometriosis, birth complicated by premature rupture of the amniotic membranes, 15% - fetal distress, 8,3% - primary and secondary weakness of labor activity, 3 women was hypotonic bleeding. According Apgar scale the results were better in the control group. Early and late postnatal complications noted the following: uterine bleeding 5% childbirth main group and 1,2% - childbirth control group, subinvolution of uterus in 8,3% and 3,6% respectively. Lohiometra occurred in 30% of patients with endometriosis and 3,6% of patients in the control group. The hospital asked about lactostasis 13,3% childbirth main group and 7,1% in the control group of women in childbirth. Conclusions. 1. Preterm labor occurred 10,4 times more in women with endometriosis than the control group. 2. Labor by Ceserean section was a third of the patients of the main group. 3. Condition of newborns of women with endometriosis were not significantly different from the condition of infants in the control group.Кафедра акушерства та гінекологі

ESTIMATION OF ANTIOVARIAN ANTIBODIES IN WOMEN WITH INFERTILITY OF UNEXPLAINED ORIGIN

Introduction. In the structure of infertile marriage the main place has still been kept by a tubal factor. The frequency of this infertility factor reaches 18-73%. Target of the study. To determine the level of antiovarian antibodies in the blood of women with tubal infertility. Materials and methods. A clinical-laboratory examination has been made of 109 women which were divided into 2 groups: Igroup (main) - 58 women with infertility of tubal origin; 2 group (control) - 51 women. The antiovarian antibodies were determined by means of immunofermental set made by the firm Bioserv Ovari-Antibodi ELISA for determination in the blood serum of autoantibodies directed against ovarian antigenes. Results. 30 women have been examined and divided in two groups in our study. The first group included 20 women with infertility of tubal origin. The second group consisted of 10 healthy women with a preserved reproductive function. The average age of women in the main group didn’t differ from the women’s age of the control group. Significant majority in the number of women with chronic salpingoophoritis in the main group (6 times more) testified to the possibility of autoimmune salpingoophoritis initiation in the group under study. The level of antiovarian antibodies was determined equal to 7,1±0,9 and in the main group - 4,1±0,5, which is 1,7 times less than in the control group. Conclusions. 1. The level of antibodies in the main group of women under study and rise of the level have been determined in patients with a secondary infertility who didn’t impregnate for more than 5 years and underwent the following surgery; tubectomy, cystectomy, adnexectomy. 2. On the contrary, the decreased level of antiovarian antibodies has been discovered in patients with a primary infertility who did not impregnate for 5 years as well as in women with uterine pathology and myoma.Кафедра акушерства та гінекологі

USAGE PHYTOMEDICATIONS IN WOMEN WITH HYPOGALACTIA

Introduction. Rational nutrition means, first of all, feeding infants during the first year of their lives with mother’s breast milk. Among numerous problems of breast feeding the most frequent one is mother’s complaint of not having sufficient amount of milk. The aim of this work was to study special features of clinical course of postnatal period and investigate the effect of "Shirafza” phytomedication on lactation in women in childbirth with a decreased lactational function. Materials and methods. 60 women in childbirth have been examined who were divided into two groups: I main group - 30 women with a decreased lactational function. II - control group - 30 women in childbirth with a preserved lactational function. Lactational function in women was evaluated on the fourth-fifth day of postnatal period on the basis of clinical examinations of the woman in childbirth, day amount of milk that is produced, food supplements of mixtures to the infant’s feeding and indeces of weight loss and its restoration during first four-five days after birth. Normal lactation was regarded a condition without food supplements, when maximum weight loss was no more than 6%, restoration of initial weight no less than 95% during early neonatal period. Results of the study and discussion. The formation of lactation during first 5 days after labor is influenced by the woman’s somatic health condition, her obstetrical-gynecologist status, obstetrical complications which were registered more frequently in women from the main group that’s why we have suggested recovery of lactational function in this group of patients by means of "Shirafza” phytomedication, stimulates all stages of lactation, prevents problems of overweight in feedings mothers and rise of glucose level in blood. Shirafza effect is conditioned by herbs in its composition. The preparation was taken by women from the main group because they complainted mostly of insufficient milk production. The preparation was-taken from the 4-th day of postnatal period by 1 capsule three times a day. 2 5 women-in labor (83,3%)were marked down to increase the volume of milk on the 5-6th day of the intakes as well as to improve milk quality which manifested inself in the absence of dry "swaddling clothes” syndrome, the baby was actively sucking mother’s breast.Кафедра акушерства та гінекологі

COURSE OF POSTNATAL PERIOD IN WOMEN WITH HYPOGALACTIA USING PHYTOMEDICATIONS

Introduction. Hypogalactia is a condition of decreased secretory activity of mammary glands in lactational period [1, c. 67; 5, c. 98]. Primary and secondary hypogalactias are distinguished. Primary one is conditioned by anatomical and functional pe¬culiarities of mammary glands as well as by moth¬er’s severe diseases associated with extragenital pathology (diabetes mellitus, diffuse toxic goiter), occurs rarely, only in 4-10% of women. Later on, women-in- childbirth who took these preparations had no lactation at first hours or days after labor or lactation appeared in later periods and was insuffi¬cient [3, c. 56; 6, c. 87]. Secondary hypogalactia is mostly a temporary condition [2, c. 35; 8, c. 23]. The reason that caused hypogalactia should be found out to this aim [4, c. 45; 7, c. 56]. A special group of women can be distinguished according to the development of primary hypoga-lactia - these are the women who underwent Cesar-ean section. Early hypogalactia is singled out as to the time of its occurrence. It appears during first 10 days after labor. Late hypogalactia occurs 10 days after. The aim of this work was to study special fea-tures of clinical course of postnatal period and in-vestigate the effect of «Shirafza» phytomedication on lactation in women in childbirth with a decreased lactational function. Material and methods of the study. 60 women in childbirth have been examined who were divided into two groups: I main group - 30 women with a decreased lactational function. II - control group - 30 women in childbirth with a preserved lactation¬al function. Life history, somatic and gynecologic diseases in the history, special features of menstrual and reproductive functions, the course of pregnancy, labor and postnatal period have been studied in all women. In newborns there were determined: condi¬tion at birth, evaluation by Apgar’s scale, primary loss of body-weight and the degree of its recovery by the fourth-fifth day of life, peculiarities of the course in early postnatal adaptational period. Lactational function in women was evaluated on the fourth-fifth day of postnatal period on the basis of clinical examinations of the woman in childbirth, day amount of milk that is produced, food supple¬ments of mixtures to the infant’s feeding and in- deces of weight loss and its restoration during first four-five days after birth. Normal lactation was re¬garded a condition without food supplements, when maximum weight loss was no more than 6%, resto¬ration of initial weight no less than 95% during early neonatal period. S.N. Gaidukov’s classification has been used to evaluate the degree of insufficiency of lactational function (1999): I degree of failure was regarded milk deficiency no more than 25% from the needed quantity; II degree - 50%, III degree - up to 75%, IV degree - over 75%. Statistical processing of the data was performed on a personal computer IBM by means of statistical programme EXCEL making use of variational statistical methods. Authenticity of special features was established by means of Stu-dent’s t-criteria. Differences of indeces were regard-ed authentic at value of p<0,05. Results of the study and discussion. At the age examination it was determined that the majority of women in childbirth were from 19 till 30: in the main group - 25 (83,3%), in the control - 21 (70%) (p>0,05). Average age of women in labor didn’t differ for a fact (25,8±4,2 and 25,1±4,1 of a year correspondingly), A high frequency of somatic pa-thology has been found out: in the main group - in 22 (73,3%), in the control - in 12 (40%) of women (p<0,05). Gynecological diseases in the anamnesis are regarded a factor of risk for disorders in the re-productive system which causes hormonal distur-bances, failure of childbearing function with further complications of postnatal period and also affects formation and duration of lactation. There were gy-necological diseases in the anamnesis in 23 (76,6%) women in the main group and 11 (36,6%) (p<0,05) - in the control. At the same time menstrual function disorders were really more often in the main group - 21 (70%) womem in comparison with the control group - in 4 (13,3%) (p<0,05). In the main group in the structure of menstrual function disorders at-tention was drawn to the predominance of such nosologic forms as dysmenorrheal - in 5 (16,6%), polymenorrhea - in 2 (6,6%), irregular menses - in 12 (40%) women. Only in the main group in the an-amnesis there were revealed such disorders as late menarche - in 1 (3,3%) (p<0,05), hypomenstrual syndrome - in 1 (3,3%) patient. A normal course of postnatal period in the main group was observed in 4 women (13,3%) in the control - in 16 (53,3%). Decrease of lactational function - in 22 (73,3%) women of the main group and 4 (13,3%) in the control one, subinvolution of uterus - in 4 (13,3%) women of the main group and 1 (3,3%) woman of the control group. As subinvolution of uterus oc-curred more often in women from the main group, the factors which contributed to the appearance of this complication were analysed. In women from the main group cardiovascular diseases were regis-tered more often - in 8 (26,6%) and in the control group - 4 (13,3%). In women with a reduced lac-tational function there is a high frequency of dis-orders in menstrual cycle and salpingo-oophoritis in the anamnesis: in the main group - in 21 (70%) and 19 (63,3%); in the control group - in 4 (13,3%) and 5 (16,6%) correspondingly (p<0,05). Incompe¬tent pregnancy in the anamnesis as a factor causing hypogalactia was registered in 53,3% women from the main group. In the majority of women in labor from the main group having hypogalactia there was a thread of abortion - 14 (46,6%), gestosis - in 10 (33,3%), anemia - in 27 (40%); placental insuffi-ciency in 12 (40%), in the control group - in 16,6; 3,3;53,3 and 6,6% cases correspondingly. The most serious intranatal factors for disorders of lactational function after labor were anomalies of labor activity and blood loss over 500 ml that were registered in both groups with almost similar fre¬quency. Both in the main and control groups there weren’t found out any authentic significant perinatal factors among full-term neonates that wound influ¬ence the formation of lactation in postnatal period. The formation of lactation during first 5 days after labor is influenced by the woman’s somatic health condition, her obstetrical-gynecologist status, ob¬stetrical complications which were registered more frequently in women from the main group that’s why we have suggested recovery of lactation¬al function in this group of patients by means of «Shirafza» phytomedication, that is a natural meth-od of high quality based on vegetable raw material which due to complex effect on the woman’s organ¬ism stimulates all stages of lactation, prevents prob¬lems of overweight in feedings mothers and rise of glucose level in blood. Shirafza effect is conditioned by herbs in its composition. Fennel (Foeniculum Mill) contains the mixture of trans-anethol with fenhon, estragol and other components. Structural resemblance of anethol and catecholamine can sup¬port estrogenic activity. Dopamin has a stimulating effect on lactation, in this way anethol can cause milk production re-sponding to dopamine on the determined sections of receptors. Due to this the effect of dopamine de-creases in stimulating milk production. Biologically active substances in Fennel composition stimulate lactogenous action in feeding mothers and also re-move evidences of meteorism, improve digestion release spasms and mobility of the intestine. Caraway provides the increase of milk amount and its nutritional qualities. Trigonella manyco- loured improves bloodflow in the mammary gland, stimulates lactatation and provides the increase of nutritional value of breast milk. Common dill (anethum graveolens) has a seda-tive and soothing action, so women in childbirth are less anxious and its anti-inflammatory action dimin¬ishes the risk of mastitis occurrence. Alcaloids of common dill only increase the amount and quality of breast milk, but in interaction with other compo¬nents of «Shirafza» pass over together with moth¬er’s milk to the infant and prevent meteorism in this way and release abdominal pain (colics) in a breast¬fed infant. The preparation was taken by women from the main group because they complainted mostly of insufficient milk production. The preparation was-taken from the 4-th day of postnatal period by 1 capsule three times a day. 25 women-in labor (83,3%)were marked down to increase the volume of milk on the 5-6th day of the in¬takes as well as to improve milk quality which manifest¬ed inself in the absence of dry «swaddling clothes» syn¬drome, the baby was actively sucking mother’s breast, the woman in childbirth felt the rush of milk, the infant didn’t cry of began to gain weight actively. Conclusions. Women in childbirth show a high frequency of complications in postnatal period (73,3%), that is significantly higher than the analogous index in the control group (40%) Usage of «Shirafza» preparation contributes to the improvement of lactational function in wom¬en in childbirth already on the 5-th day of postnatal period.Кафедра акушерства та гінекологі

PECULIARITIES IN THE COURSE OF POSTNATAL PERIOD IN WOMEN WITH HYPOGALACTIA

Introduction. Rational nutrition means, first of all, feeding infants during the first year of their lives with mother’s breast milk. The aim of this work was to study special features of clinical course of postnatal period in women with hypogalactia. Material and methods of the study. 60 women in childbirth have been examined who were divided into two groups: I main group - 30 women with a decreased lactational function. II - control group - 30 women in childbirth with a preserved lactational function. S.N. Gaidukov’s classification has been used to evaluate the degree of insufficiency of lactational function (1999). Anamnesis data, extragenital diseases, complications in the courseof pregnancy, labor and postnatal period were taken into account at making a diagnosis of oligogalactia function. Statistical processing of the data was performed on a personal computer IBM by means of statistical programme EXCEL making use of variational statistical methods. Authenticity of special features was established by means of Student’s t-criteria. Differences of indeces were regarded authentic at value of p<0,05. Results of the study and discussion. At the age examination it was determined that the majority of women in childbirth were from 19 till 30: in the main group - 25 (83,3%), in the control - 21 (70%) (p>0,05). An uncomplicated course of postnatal period was registered significantly more seldom in the main group of lying-in women - in 4 (1 3,3%)(p<0,05) than in the control group - in 16 (53,3%). A normal course of postnatal period in the main group was observed in 4 women (13,3%) in the control - in 16 (53,3%).Кафедра акушерства та гінекологі

THE INFLUENCE OF PYELONEPHRITIS COMPLICATIONS ON COURSE OF PREGNANCY AND LABOR

Pyelonephritis - nonspecific, infectious-inflammatory process with primary and primary lesion interstitial tissue walls and renal tubules, with subsequent involvement of glomerular and vascular system. Different pathological conditions, which provoked acute stage of chronical pyelonephritis among pregnant women has been analized. Among: anomalies of kidney development, glomerulonephritis, hypertonic disease, preeclampsia and oth. According retrospective investigation of case history of women of postpartum period which have complicated pyelonephritis of Chernivtsi region has been received such characteristics: general amount of cases of nephrological complicated diseases during 2010-2013 consists 87; ratio of these diseases consists: pyelonephritis - 78,16%, glomerulonephritis - 9,1% and oth. - 12,74%. Most part of all background conditions, which contributes development of acute stage of chronical pyelonephritis compound anomalies of kidney development (51%), among them the most - single left kidney (34%). Different groups of exciters of chronical pyelonephritis during pregnancy has been analized: Ar. Piogenes, Streptococcus spp., E. coli, Candida albicans, St. aureus, Enterobacter aerogenus, S. Haemolyticus, S. epidermidis. Negative results of bacteriological examination of urine after finishing course of antibioticotherapy has been founded efficacy of antibioticotherapy in pregnant women with complicated pyelonephritis. The disappearance or significant reduction in the severity of the clinical manifestations of the disease: negative Pasternatsky symptom, disappearance of pain in the lumbar region, the normalization of body temperature and reduce the intensity of other general clinical symptoms.Кафедра акушерства та гінекологі

A ROLE OF OVARIAN AUTOIMMUNITY IN THE PATHPHYSIOLOGY OF PREMATURE OVARIAN INSUFFICIENCY

Premature ovarian failure (POF) is a term usually used to describe women younger than 40 years of age who present with amenorrhea. Diagnosis of POF is on the basis of follicle stimulating hormone level in the menopausal range associated with amenorrhea before the age of 40. Women diagnosed with the POF suffered from anovulation and hypoestrogenism and presented with primary or secondary' amenorrhea, infertility, sex steroid deficiency, and elevated gonadotrophins [1, p. 245] POF is the causative factor in 10 28 % of women presenting with amenonhea and in 4 18 % with secondary amenorrhea [2, p. 236; 3, p. 45]. The course of POF is poorly defined [4, p. 112]. Perhaps 50 % of spontaneously affected woman have the evidence of follicular activity and probably 25 % ovulate even before the diagnosis is established. Some of these women even conceive. It is increasingly felt that it may be appropriate to refer to them as patients with «primary ovarian insufficiency.» The term primary ovarian insufficiency (POI) originally suggested by Albright appropriately describes a continuation of impaired ovarian function and is also less stigmatizing than the terms used previously [5, p. 38]. Two mechanisms are probably involved in POL namely follicle dysfunction and follicle depletion [6, p. 253]. The existing follicles in the ovary, in follicle dysfunction, do not function normally due to some pathological process such as for e.g. FSIT receptor mutation [7, p. 86J. On the oilier hand, in women with follicle depletion there are no primordial follicles probably due to inadequate initial pool of primordial follicles or destruction of follicles due to toxins or autoimmune mechanisms ]5, p. 39]. Lhe primary goal of the scientists working in this area should be to focus on the etiology of lA)! [8, p. 99]. Some of the causes can lead to complete absence of oocytes, and others can lead to inability of follicles to mature or to disordered folliculogenesis. The causes could be chromosomal, genetic, autoimmune, metabolic (galactosemia), infection (mumps), and iatrogenic [9, p. 123]. Among the several mechanisms that account for the pathogenesis of spontaneous POL genetic and autoimmune mechanisms play a major role. X chromosome abnormalities have been systematically reviewed by Persani ct al [10, p. 96]. Nomial ovarian function requires two functioning X chromosomes. In the Turner Syndrome, there is a complete loss of the second chromosome resulting in the most severe and irreversible POl often clinically evident prior to menarche [11, p. 176]. Prevalence of other genetic defects causing POI has been difficult to determine. Gene for blepharophimosis/ptosis/POI syndrome has been recently reported but has not been seen commonly in patients with POl. Of the various knock out mice models created with deficient ovarian function, the most interesting one is a heterozy gous FSH receptor knock out mouse, which has exhibited a reduced follicle reserve and early ovarian depletion [11, p. 29]. hi this review, we will focus on the role of autoimmunity in the pathophysiology of POI. The human ovary can be a target for an autoimmune attack under various circumstances. Clinically, the ensuing ovarian dysfunction often results in premature ovarian insufficiency characterized by amenorrhea lasting 4 6 months and is classically defined as secondary amenorrhea accompanied by a hypergonadotropic—hypoestrogenic condition before die age of 40 years [12, p. 49]. It has long been recognized that POI could be associated with nearly all organ specific and non- organ- specific autoimmune diseases, and its association with the endocrine glands such as the thyroid, pancreas, and adrenal glands has been reported [13, p. 68]. Vallotlon and Forbes [ 14, p.66] were the first to describe the presence of antibodies to rabbit ova cytoplasm using sera from POI patients. Autoimmune POI (Al POI) is characterized by organ specific targeting of the immune response accompanied by tissue destruction, which can have widespread systemic complications in severe cases. The disease affects 1 % of the general population [15, p. 781). There could be several reasons for the differences among the study results. First, study design elements, such as antibody test format and antigen preparation and criteria for study and comparison groups differ. Second, there may be several antigenic targets, and often only one may have been assessed. Moncayo et al [19, p. 581. developed an ELISA using microsomes from bovine corpora lutea as the antigen. Luborsky et al [17, p. 39]. developed an ELISA kit using total human ovary /oocytes as antigen and showed that sera from 71 % of women with POF in their study had antibodies either to whole ovary or to oocytes. We also propose that the AOA test should be a part of the battery of tests included for infertility treatment and management. Very little is known about the precise nature of the ovarian antigens that are recognized by the antibodies in sera. Antigens of oocyte, [14, p. 238) corpus luteum [17, p. 69], granulosa cells 116, p. 128], and ZP [21, p. 62] have been reported to act as autoantigens; however, their molecular identity and pathophysiologic significance remain obscure. The oocyte seems to be the most often targeted cell of AOA detected in cases of ovarian diseases as well as in women with poor assisted reproductive technologies (ART) outcomes [22, p. 89]. A thorough literature review on infertility with autoimmune involvement has indicated that very few proteins have been formally identified and characterized using sera of women with infertility [28, p. 146]. One report demonstrated autoantibodies to a enolase, [30, p. 66] and several reports have shown the presence of circulating antibodies directed toward different ovarian structures [16, p. 49; 17, p. 57; 22, p. 86] Identification and systematic characterization of target antigens are the prerequisites for elucidation of the underlying immunologic mechanisms and also for devising better approaches for the diagnosis and treatment of POI leading to infertility 136, p. 89]. (>nce their identity has been established, they could be used for simple, noninvasive diagnostic tests to screen large populations of women with infertility or repeated implantation failures as well as to screen patients before and after enrollment in an IVF ET program. Although a number of tests have been developed to detect these antibodies, neither their specificity nor their diagnostic relevance has been established.Кафедра акушерства і гінекологі

PRO APOPTOTIC PEPTIDES AS POTENTIAL THERAPY FOR PERITONEAL ENDOMETRIOSIS

Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with non¬steroidal anti inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated zl3, using phage display. As most endometri¬osis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the zl3 receptor as the cyclic nucleotide-gated channel (13, a sorting pathway protein. We then linked zl3 with an apoptosis inducing peptide and with an endosome -escaping peptide. When these peptides were co- administered into the peritoneum of baboons with endometri¬osis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal en¬dometriosis in humans. Endometriosis occurs only in higher primates, including hu¬mans and baboons. Spontaneous endometriosis reportedly occurs in about 25 % of captive baboons, and its prevalence increases with captivity duration [5]. Experi¬ments in non -human primates show a clear positive correlation between endometrio¬sis and a diet containing the chemical dioxin [6, 7], which may promote endometrio¬sis by acting as an estrogen like factor, hi patients with endometriosis, many of the pathologic processes including inflammation, the immune response, angiogenesis and apoptosis are all favoured for promoting endometriosis in a manner dependent on steroid hormones - |X, 9|. Thus, in the past, most treatment was via therapeutics¬targeting steroid hormones and their receptors [10, 11 j. These drags can only be administered for a short term due to side effects. Current first-line therapy is oral con-traceptive pills, which halt an ovulation and suppress endometriosis tissue growth with minimum side effect [ 12 j. Oral contraceptive pills are also administered with nonsteroidal anti-inflammatoiy drags, further reducing endometriosis-associated pain 113, 14]. Nonetheless, these treatments do not remove endometriosis, and patients with severe symptoms must undergo surgery. However, even then symptoms can re¬cur, requiring multiple surgeries in many cases [15]. Thus, it is critical to develop new strategies to cure this disease, particularly to prevent recurrence after surgery. One proposed cause for endometriosis is through retrograde menstrual reflux of the endometrium that becomes implanted in regions of the pelvis [16], most common¬ly on the ovaries and areas facing the peritoneum [17, 18]. Thus, we hypothesized that a molecule specifically expressed on the endometrial surface could be targeted by a peritoneally injected drug if that marker is not expressed by other peritoneal sur¬faces. We report here the identification of such a reagent, namely, a 9-mer peptide that specifically binds to glandular epithelial cells of endometriosis in peritoneum. We also identified its receptor as the cyclic nucleotide-gated channel [)39(CNGB3) and confirmed its expression in endometriosis. On the basis of these findings, we de¬veloped potential therapeutics for endometriosis targeting disease lesions at peritone¬al surfaces mid tested their activity in baboon endometriosis models. This study pre¬sents a strategy that could be useful to treat peritoneal endometriosis in humans. Identification of peptides targeting peritoneal endometriosis, As yet, there is neither an in vitro model nor an in vivo animal model available to study endometriosis, other than non-human primates. To identify a peptide that specifically binds to epithelial cells in endometriosis, we hypothesized that some, if not all, human endometrial adenocarcinoma cell lines would express cell surface pro¬teins expressed in glandular epithelial cells in endometriosis. To devise a probe that specifically binds to the endometriosis surface but not to the surface of other organs facing the peritoneum, we undertook subtractive phage library.' screening [ 18, 19|. A T7 phage-based library (109clones, 101 ‘plaque-forming unit) of linear 9-mer peptides was injected into tire peritoneal cavity of a female mouse to allow absorption of phage clones to the peritoneal surface in vivo for 1 h. The precleared libraiy was re¬covered from peritoneal fluid and added to a monolayer of human endometrial ade¬nocarcinoma Ishikawa cells cultured in vitro. We choose Ishikawa cells as this cell line shares characteristics with mature endometrial epithelial cells [20, 21]. We also wanted to identify a peptide internalized by endometrial glandular epithelial cells so that a dnig conjugated with that peptide would penetrate target cells. We therefore incubated phage with live Ishikawa cells at 37 °C for 30 min to facilitate phage inter¬nalization. Selected phage clones were recovered after solubilizing cells with deter¬gent and amplified in bacteria. After three rounds of subtractive library screening, the number of phage clones with Ishikawa cell- binding activity relative to the total num¬ber of added phage increased 10,000-fold. The third screen-positive phage pool was overlayed on frozen tissue sections of endometriosis lesions surgically isolated from endometriosis patients. Immunohisto- chemislry using an anti phage antibody showed positive signals on glandular epithe¬lial cells, particularly at apical cell surfaces in endometriosis. These results suggest that, despite differences between endometriosis and endometrial adenocarcinoma, the positive phage pool bound to the Ishikawa cancer cell line contained a clone or clones binding to endometrial glandular epithelial cells. Sequencing of insert DNAs from isolated phage clones revealed the deduced consensus sequence VRRAXNXPG (where X represents a varying amino-acid residue,. The presence of the consensus sequence attests to the high specificity of selected clones. In vitro binding assays in¬dicated that each clone bound to Ishikawa cells at higher efficiency than to control skin epidermoid carcinoma A431 cells. Among the clones, zl3, which displayed the sequence VRRADNRPG, was the strongest binder. Z13 phage bound to endometrial adenocarcinoma SNG--II. RL95-2 and HeclA cells but not to 431, prostate cancer PC3 or cervical cancer HeLa cell. To confirm zl3 peptide-binding activity, we chemically synthesized zl3 peptide with an ammo-terminal lluorcscein isothiocyanate (FITC) tag and added to Ishikawa cells and control A431 cells . Fluorescence micrographs showed binding of FITC-zl3 to Ishikawa cells, but not to A431 cells. Micrograph of Ishikawa cells showed a punc¬tate cytoplasmic staining patteni, suggesting that zl3 is internalized to endosomes. Fluorescence micrographs of Ishikawa cells (left) and control A431 cells (nght) overlayed with a synthetic z.13 peptide tagged with fluorescein isothiocyanate (FITC) and left at 37 °C for 15 min. Scale bar, 50 pm. Visualization and isolation of the zl3 peptide receptor. Left: cell surface proteins expressed on Ishikawa cells were bioti¬nylated. Cell lysates were bound to z 13 peptide- conjugated agarose beads, and bound proteins were eluted with irrelevant peptide (lane 1) or zl3 peptide (lane 2). Biotinyl¬ated proteins in each eluate were detected by peroxidase- conjugated avidin and a lu¬minescent peroxidase substrate. Right: silver staining of peptide affinity purified zl3 receptor from endometriosis. Endometriosis tissues isolated from patients were ho¬mogenized, and microsome membrane fraction was prepared. Proteins solubilized with detergent were applied to a zl3 peptide conjugated agarose column, and bound proteins were eluted with irrelevant peptide (lane 1) or zl.3 peptide (lane 2). Proteins in each eluate in SDS-PAGE were detected by silver staining, (c) Fluorescence mi¬crographs of HeLa cells transfected with control empty vector (upper row) or with an expression vector encoding CNGB3 MYC (lower row). Binding of FlTC-z.13 pep¬tide (green) to HeLa cells transfected with mammalian expression vectors (a,d), im- munostained with anti-MYC followed by Alexa 549 (red)-conjugated anti-mouse IgG antibody (b,e) and merged images including 4',6-diamidino-2 phenylindole (blue) to indicate nuclear staining (c.f). Scale bar, 20 urn. Identification of the zl.3 peptide receptor, To develop a clinically relevant therapeutic strategy, we searched for the /13 peptide receptor. To do so, Ishikawa cells were surface biotinylated and lysed, and then lysates were incubated with z 13 peptide-conjugated agarose beads. Bead bound materials were then eluted by zl3 peptide, and biotinylated proteins detected by avi- din blot, revealing a single 68-kDa protein. To identify this protein, the microsomal membrane fraction was prepared from endometriosis tissue surgically removed from patients. Membrane proteins solubilized with detergent were applied to a zl 3 peptide affinity column and column-bound materials were eluted by zl3 peptide. A silver- stained gel revealed a 68-kDa protein, and proleomic analysis identified the peptide sequence, QRTALYK, which is unique to the CNGB3 GNGB3 protein has six trans¬membrane domain [22]. A large part of this protein is buncd in the lipid bilayer, the N and C-terminal domains are cytoplasmic and presumably the zl3-binding regions are extracellular. To assess binding of FITC-zl3 to CNGB3, we transfected HeLa cells with a mammalian expression vector encoding a GNGB3—MYC fusion protein or with control empty vector. FITG-z.13 did not bind to vector transfected control cells. HeLa cells transfected with the CNGB3--MYG expression construct and stained by anti-MYC antibody showed an endosome-like pattern . When FITC-zl3 was add¬ed to culture medium of CNGB3 -MYC-expressing HeLa cells, FITC-zl3 bound to sites marked by MYC epitope expression. The kinetics of FITC-zl3 binding to CNGB3 MYC expressing HEK293T cells showed a A'd of 9.759 x 10 11M and aB- max-of 0,9086 moles per receptor. These results indicate that recombinant CNGB3 ex¬pressed in a mammalian cell has zl 3 peptide-binding activity on the cell surface and internalized to endosome, supporting the hypothesis that GNGB3 is the zl3 recep- tor.To examine CNGB3 protein expression in endometriosis, we generated a mouse monoclonal antibody against a peptide sequence of human CNGB3, corresponding to K [12, 14, 151 to P [1, 3] within the cytoplasmic domain. Antibody specificity was validated by immunostaining of CNGB3—MYG expressed in HeLa cells. This anti¬body robustly stained glandular epithclia of endometriosis in tissue sections. When we evaluated endometriosis tissue sections from 35 endometriosis patients, we found that 31 specimens showed strongly positive immunostaining and 4 showed weak/negative immunostaining. Peritoneal surfaces from cycling women without en¬dometriosis were not stained by this antibody, whereas those from endometriosis pa¬tients were stained by this antibody, suggesting that endometrial cells are spread across a wide area on the peritoneum of endometriosis patients. Eutopic endometrial tissues at secretory and proliferative phases were weakly stained by this antibody. Immunohistochemistry of human tissues showed that this antibody did not stain the surface of organs facing the peritoneal cavity (data not shown). Histology of hematoxylin and eosin-stained tissue sections revealed evidence of endometriosis in all three animals treated with a mixture of dKLAK- /13 and 1ILA11 zl3 peptides. Terminal deoxynucleotidyl transferase mediated dUTP nick end label¬ing (TUNEL) assays of endometriosis lesions collected from three untreated control baboons revealed no TUNEL positivity in gland tissue. By contrast, we found TUNEL positive glands in tissues collected from all three baboons treated with the dKLAK zl3 and HLAlT zl3 peptide mixture, those signals were seen in glandular epithelial cells in ovarian endometriosis and in the lumen of endometrial gland in the omentum. No evidence of apoptosis was detected in cells facing peritoneum in liver, kidney, spleen, colon and stomach (data not shown) These results show overall, as proof of concept, that /13 targeted induced apoptosis occurred in endometriosis mod¬el in baboons in vivo. A general strategy of translational research is to identity a gene product associ¬ated with a disease of interest and then develop drug targeting that molecule. Howev¬er, in identifying peptides that target endometriosis but not non endometrial cells lac¬ing the peritoneal cavity, we took a novel approach that does not require knowledge of a target We also demonstrated that CNGB3 is the receptor for zl3 peptide based on its expression pattern, not its function. We feel that analysing CNGB3 function in detail is not a priority' for clinical application of this work.Кафедра акушерства і гінекологі