Ovarian Estrogen Synthesis Reduced by Peritoneal Endometriosis in Rat Autologous Transplantation Model

OBJECTIVE: To investigate the effects of peritoneal endometriosis on rat ovaries. METHODS: A rat model of peritoneal endometriosis was established by autologous transplantation. qPCR was performed to measure mRNA levels of steroid hormone and steroid synthesis-related genes in the ovaries of endometriosis rats. Immunohistochemistry was performed to characterize the distribution of FSHR in the ovaries of endometriosis rats. RNAseq was performed to find pathological changes in the ovaries of endometriosis rats. RESULTS: By qPCR, it was revealed that mRNA levels of steroid hormone synthesis-related genes were decreased in the ovaries of rats with endometriosis; With IHC, observed that FSHR expression was significantly decreased in the antral follicles of rats with endometriosis. RNAseq revealed that endometriosis affected transcription of the genes related to the microtubule structure and tight junctions of rat ovarian cells. CONCLUSION: Peritoneal endometriosis decreased the genic expression of ovarian steroid hormone synthetases and FSHR protein level in granulosa cells of antral follicles, and reduced the mRNA levels of the microtubule structure and tight junctions in rat ovarian cells, which contribute to the impairment of ovarian function.

Folliculogenesis, Fertility and Biotechnology in Dairy Cattle

The ovarian follicle population is formed by thousands of follicles, preantral and antral, where oocytes are included. During fetal life, the first follicles produced are preantral, and, as they undergo the development process, they reach the final stage of antral follicles, where a cavity/or antrum is developed. All this growth phase is called folliculogenesis, and this chapter will abord the most important aspects of this process. Moreover, not all follicles reach the preovulatory phase and can be fertilized, so we will discuss how reproductive biotechniques can positively influence the fertility of bovine females. We will also discuss the possibility of antral follicle count to influence reproductive performance and the correlation to biotechniques. Finally, we present alternatives on how to improve fertility and productive efficiency in dairy herds.

CONNECTION OF VITAMIN D WITH MARKERS OF REPRODUCTIVE FUNCTION IN PATIENTS WITH WITH POLYCYSTIC OVARY SYNDROME

Objective. To investigate the relationship between vitamin D and markers of reproductive function in women with polycystic ovary syndrome (PCOS) and to assess of their changes with cholecalciferol.Materials and methods. Thirty patients with PCOS and 20 healthy women with vitamin D deficiency, who similar for the age and body weight, were examined. Vitamin D, antimullerian hormone (AMH), number of antral follicles, follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2), sex hormone-binding globulin (SHBG), ovarian ultrasound parameters before and after 12 weeks of cholecalciferol therapy at a dose of 4000 IU daily were determined.Results. Before treatment, was not found association between vitamin D and AMG and the number of antral follicles in both groups. In patients with PCOS was found a negative relationship between vitamin D and T (r = – 0.579; P < 0.001), free androgen index (r = – 503; Р < 0.01), LH/FSH (r = – 0.591, P < 0.001), T/E2 (r = – 0.603; P < 0.001) and positive correlation with SHBG (r = 0.611; P < 0.001), which indicates the role of vitamin D deficiency in the formation of hyperandrogenemia. The therapy of cholecalciferol did not affect the level of AMН, at the same time, was accompanied by changes in gonadotropin secretion and their ratio, reduction of hyperandrogenemia and positive dynamics of folliculogenesis.Conclusion. We believe that the appointment of vitamin D in patients with PCOS is promising, as it has a positive effect on indicators that reflect the state of reproductive function.

Comparative characteristics of patients with infertility when applying melatonin in complex preparation for assisted reproductive technologies

Оbjective of the study was to conduct a retrospective comparative characterization of patients with infertility who took or did not take melatonin with assisted reproductive technologies (ART).Materials and methods. In our study, we examined 89 women. The first (control) group included 13 healthy women oocyte donors who got pregnant on their own and gave birth to their own healthy children, the second group - 33 patients with infertility, who took 3 mg of the preparation "Vita-melatonin" produced by "Kyiv Vitamin Plant" at the same time before bedtime, two weeks before and during ovulation stimulation, the third group - 43 patients with infertility who did not take melatonin preparation before and during ovulation stimulation. There were no women who worked night shifts among the patients. Medical documentation of women of the control group and those with infertility, data of gynecological, ultrasound examination, hormones blood were analyzed. Ultrasound examination of the pelvic organs was performed on all patients with the device "Mindray DC-80 X-Insight", and measurements were performed using a transvaginal sensor. The thickness and structure of the endometrium were evaluated, and the number of antral follicles (NAF) ranging in size from 2 to 10 mm was counted in each ovary. All patients were tested for serum levels of the anti-mullerian hormone (AMG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), prolactin (PRL), progesterone (P), thyroid-stimulating hormone (TSH), triiodothyronine (T4).Results. The average age of women in the first (control) group was 27.08 ± 12.38 years, the second (taking melatonin) - 33.12 ± 8.18 years, the third (not taking melatonin) - 30.95 ± 7.07 years > 0.05), i. e. the age of the patients of the examined groups was equal. It should be noted that in the studied patients of both groups, the occurrence of primary infertility exceeded secondary infertility 2.7 times in the second group (p < 0.05) and 1.7 times in the third (p < 0.05). Infertility factors such as reduced ovarian reserve, habitual miscarriage and infertility of unknown origin were more common in patients of the second group, and endometriosis, tubal factor and male factor in the third, although the difference was not significant. The available extragenital pathology did not differ in the patients of the examined groups. The number of antral follicles was significantly higher in both ovaries of women in the control group compared with patients of the second and third groups. While the thickness of the endometrium did not differ significantly in groups, although in women of the control group it was slightly less. Regarding the study of hormonal status, it should be noted that we did not find a significant difference in the levels of hormones in the blood of women we examined. Exceptionally, there was a significant difference (p < 0.001) in progesterone content between the second (0.62 ± 0.052 nmol/l) and third (181.63 ± 13.87 nmol/l) groups. Also, the patients of the third group had significantly (p < 0.05) higher levels of FSH in blood (8.25 ± 0.63 mUn/ml) compared with the control group (4.93 ± 0.69 mUn/ml).Conclusions. The examined women in the control group, as well as infertility patients who received melatonin two weeks before the expected menstruation and during ovulation stimulation, and infertility patients who did not receive this preparation in similar programs, did not differ in age, occurrence of primary and secondary infertility, the factor that led to infertility, concomitant extragenital pathology, ovarian reserve and hormone levels of the reproductive panel. That is, they were equal in our study.

Anti-Müllerian Hormone in Pathogenesis, Diagnostic and Treatment of PCOS

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-aged women. It is characterized by chronic anovulation, hyperandrogenism, and the presence of polycystic ovary in ultrasound examination. PCOS is specified by an increased number of follicles at all growing stages, mainly seen in the preantral and small antral follicles and an increased serum level of Anti-Müllerian Hormone (AMH). Because of the strong correlation between circulating AMH levels and antral follicle count on ultrasound, Anti-Müllerian Hormone has been proposed as an alternative marker of ovulatory dysfunction in PCOS. However, the results from the current literature are not homogeneous, and the specific threshold of AMH in PCOS and PCOM is, therefore, very challenging. This review aims to update the current knowledge about AMH, the pathophysiology of AMH in the pathogenesis of PCOS, and the role of Anti-Müllerian Hormone in the treatment of this syndrome.

Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

Single-Cell Transcriptomics Analysis of Human Small Antral Follicles

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand–receptor interactions regarding the transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.

Zika virus infection in the ovary induces continuously elevated progesterone level and compromises conception in interferon α/β receptor-deficient mice

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impacts of ZIKV infection on nonpregnant female reproductive health are not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/β receptor-deficient ( Ifnar1 -/- ) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle, and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months post infection, the damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE ZIKV, a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barre syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on genital tract of non-pregnant female. This study investigated the effects of ZIKV on the ovary in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function, and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.