Mature Follicles Research Articles

The insulin-like growth factor 1 stimulates ovarian steroidogenesis and oocyte maturation in spotted steed Hemibarbus maculatus

The insulin-like growth factor 1 (IGF1) plays important roles in fish reproduction. To understand the role of IGF1 in steroidogenesis and oocyte maturation, we investigated expression pattern and functions of Igf1 during in vitro maturation of spotted steed (Hemibarbus maculatus) oocytes. Igf1 expression quantified by qPCR and western blotting revealed that both Igf1 mRNA and protein was significantly increased in mature follicles. The immunofluorescence analyses showed IGF1 protein signal was detected in the theca cells of ovarian follicles during vitellogenesis stage, and in both theca cells and granulosa cells of matured oocytes. Incubation of ovarian follicles with recombinant IGF1 (rIGF1) significantly enhanced the rate of spotted steed oocyte germinal vesicle breakdown (GVBD), a marker of oocyte maturation. qPCR analysis showed that the transcription of steroidogenesis-related genes (3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a) were influenced after IGF1 treatment. In addition, the expression profiles of receptors of Igf1, Fsh and Lh indicated that a cross talk response to IGF1 induced oocyte maturation. Furthermore, IGF1 treatment did not affect ERK protein expression but significantly upregulated CyclinB and AKT protein expression. All together, these findings help us to understand the molecular mechanism of IGF1 in regulation ovarian steroidogenesis and oocyte maturation of spotted steed.

Search performance and octopamine neuronal signaling mediate parasitoid induced changes in Drosophila oviposition behavior

Making the appropriate responses to predation risk is essential for the survival of an organism; however, the underlying mechanisms are still largely unknown. Here, we find that Drosophila has evolved an adaptive strategy to manage the threat from its parasitoid wasp by manipulating the oviposition behavior. Through perception of the differences in host search performance of wasps, Drosophila is able to recognize younger wasps as a higher level of threat and consequently depress the oviposition. We further show that this antiparasitoid behavior is mediated by the regulation of the expression of Tdc2 and Tβh in the ventral nerve cord via LC4 visual projection neurons, which in turn leads to the dramatic reduction in octopamine and the resulting dysfunction of mature follicle trimming and rupture. Our study uncovers a detailed mechanism underlying the defensive behavior in insects that may advance our understanding of predator avoidance in animals.

Transcriptomic diversification of granulosa cells during follicular development between White Leghorn and Silky Fowl hens

Egg production rate in chicken is related to the continuity of follicle development. In this study, we found that the numbers of white prehierarchical, dominant, and yellow preovulatory follicles in the high-yielding layer breed, White Leghorn (WL), were significantly higher than those in the low egg-yielding variety, Silky Fowl (SF). The proliferation and differentiation of granulosa cells (GCs) play an important role in follicle maturation. Histological observation revealed a large number of melanocytes in the outer granulosa layer of follicles in SF but not in WL. Finally, RNA-sequencing was used to analyze the gene expression profiles and pathways of the GC layer in the follicles in both WL and SF hens. Transcriptome analysis of prehierarchical GCs (phGCs) and preovulatory GCs (poGCs) between WL and SF showed that steroid hormone-, oxytocin synthesis-, tight junction-, and endocytosis-related genes were expressed at higher levels in WL phGCs than in SF phGCs, whereas the insulin signaling pathway- and vascular smooth muscle contraction-related genes were upregulated in SF phGCs. Fatty acid synthesis, calcium signaling, and Wnt signaling pathway-related genes were expressed at higher levels in WL poGCs than in SF poGCs; however, adrenergic signaling, cGMP-PKG, and melanogenesis-related genes were upregulated in SF poGCs. These results indicate that genes that promote GC proliferation and secretion of various sex hormones are more active in WL than in SF hens. The upregulated signaling pathways in SF help in providing energy to GCs and for angiogenesis and melanogenesis. In vitro experiments confirmed that both the proliferation of poGCs and synthesis of reproductive hormones were higher in WL than in SF hens.

Lef1 and Dlx3 May Facilitate the Maturation of Secondary Hair Follicles in the Skin of Gansu Alpine Merino.

Lymphatic enhancer factor 1 (Lef1) and distal-less homeobox 3 (Dlx3) are the transcription factors involved in regulating hair follicle development in mice, goats, and other animals. Their deletion can lead to hair follicle deficiency. In this study, hematoxylin–eosin staining (HE), real-time quantitative PCR (RT-qPCR), immunohistochemistry, and immunofluorescence were used to analyze the expression, location, and biological functions of Lef1 and Dlx3 in the lateral skin of Gansu Alpine Merino aged 1, 30, 60, and 90 days. The results revealed that the number of hair follicles decreased with age and was significantly higher at 1 day than in the other three age groups (p < 0.05). The mRNA levels of Lef1 and Dlx3 in the skin of 30-day old Gansu Alpine Merino were significantly higher than those in the other three age groups (p < 0.05). Protein expression of Lef1 and Dlx3 was lowest at 1 day (p < 0.05) and peaked at 60 days. Lef1 and Dlx3 exhibited a high density and strong positive expression in the dermal papillae; additionally, Dlx3 exhibited a high density and strong positive expression in the inner and outer root sheaths. Collectively, Lef1 and Dlx3 may facilitate the maturation of secondary hair follicles, which is mainly achieved through the dermal papillae and inner and outer root sheaths.

Expression of voltage-gated Ca2+ channels, Insp3Rs, and RyRs in the immature mouse ovary

BackgroundThe postnatal mammalian ovary undergoes a series of changes to ensure the maturation of sufficient follicles to support ovulation and fecundation over the reproductive life. It is well known that intracellular [Ca2+]i signals are necessary for ovulation, fertilization, and egg activation. However, we lack detailed knowledge of the molecular identity, cellular distribution, and functional role of Ca2+ channels expressed during folliculogenesis. In the neonatal period, ovarian maturation is controlled by protein growth factors released from the oocyte and granulosa cells. Conversely, during the early infantile period, maturation becomes gonadotropin-dependent and is controlled by granulosa and theca cells. The significance of intracellular Ca2+ signaling in folliculogenesis is supported by the observation that mice lacking the expression of Ca2+/calmodulin-dependent kinase IV in granulosa cells suffer abnormal follicular development and impaired fertility.ResultsUsing immunofluorescence in frozen ovarian sections and confocal microscopy, we assessed the expression of high-voltage activated Ca2+ channel alpha subunits and InsP3 and ryanodine receptors in the postnatal period from 3 to 16 days. During the neonatal stage, oocytes from primordial and primary follicles show high expression of various Ca2+-selective channels, with granulosa and stroma cells expressing significantly less. These channels are likely involved in supporting Ca2+-dependent secretion of peptide growth factors. In contrast, during the early and late infantile periods, Ca2+ channel expression in the oocyte diminishes, increasing significantly in the granulosa and particularly in immature theca cells surrounding secondary follicles.ConclusionsThe developmental switch of Ca2+ channel expression from the oocytes to the perifollicular cells likely reflects the vanishing role of the oocytes once granulosa and theca cells take control of folliculogenesis in response to gonadotropins acting on their receptors.

Reproductive toxicity of long-term exposure to environmental relevant concentrations of cyprodinil in female zebrafish (Danio rerio)

In recent years, the widespread use of the pesticide cyprodinil has attracted attention due to its harmful effects on aquatic organisms. The purpose of this study was to evaluate the adverse effects of long-term exposure to cyprodinil on the reproductive system of female zebrafish. After the embryos had been treated with 0.1, 1 and 10 μg/L cyprodinil for 180 days, we observed that female fish treated with 1 and 10 μg/L cyprodinil showed decreased sexual attractiveness, a decreased proportion of primordial follicles in the ovary, an increased proportion of mature follicles, and increased egg production. Moreover, exposed females that mated with normal males produced offspring with increased rates of mortality and deformity (the F1 generation). In addition, the levels of gonadotropin and testosterone (T) were increased in females after cyprodinil exposure, especially in the 10 μg/L treated group. After cyprodinil treatment, some key genes in the hypothalamic-pituitary-gonad axis underwent significant changes. For example, gene expression of brain gonadotropin-releasing hormone receptors (gnrhr1, gnrhr2 and gnrhr4) was significantly downregulated after cyprodinil treatment. The study found that expression of the aromatase (cytochrome P450 family 19 subfamily A polypeptide 1a, cyp19a1a) responsible for converting T into estradiol was significantly downregulated after cyprodinil treatment, consistent with elevated T levels in the ovaries and muscles. In summary, these data provide a more comprehensive understanding of the toxicity of cyprodinil and may inform evaluation of the ecotoxicity of cyprodinil to female reproduction at environmentally relevant concentrations.

Arsenic, Oxidative Stress and Reproductive System.

Infertility is a severe medical problem and is considered a serious global public health issue affecting a large proportion of humanity. Oxidative stress is one of the most crucial factors involved in infertility. Recent studies indicate that the overproduction of reactive oxygen species (ROS) or reactive nitrogen species (RNS) may cause damage to the male and female reproductive systems leading to infertility. Low amounts of ROS and RNS are essential for the normal functioning of the male and female reproductive systems, such as sperm motility, acrosome reaction, interactions with oocytes, ovulation, and the maturation of follicles. Environmental factors such as heavy metals can cause reproductive dysfunction in men and women through the overproduction of ROS and RNS. It is suggested that oxidative stress caused by arsenic is associated with male and female reproductive disorders such as through the alteration in sperm counts and motility, decreased sex hormones, dysfunction of the testis and ovary, as well as damage to the processes of spermatogenesis and oogenesis. This review paper highlights the relationship between arsenic-induced oxidative stress and the prevalence of infertility, with detailed explanations of potential underlying mechanisms.

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in InVitro-Matured Oocytes.

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.

Integral role of BMP-15 in premature ovarian insufficiency

Introduction: Premature ovarian insufficiency (POI) being a heterogeneous genetic disease involves the interaction of multiple genes and environmental factors and has been associated with several chromosomal abnormalities, single gene mutations, and genetic polymorphisms. BMP-15 (bone morphogenic protien-15) is a member of the transforming growth factor β (TGF-β) family. BMP-15 gene product (protein) has 3 domains, mature domain (c-terminal region) of BMP-15 protien binds to receptors located on the granulosa cell surface to participate in key steps regarding ovarian function, such as granulosa cell proliferation and follicle maturation, ovulation rate modulation, oocyte competence determination and regulating granulosa cell sensitivity to FSH (Follicle stimulating hormone). Single nucleotide polymorphisms (SNPs) of the BMP-15 gene are associated with POI. Materials &amp; Methods: 30 POI patients and 30 healthy age matched controls were recruited for cytogenetic and molecular analysis in this case-control study. 10 ml of whole blood was collected for karyotyping and Molecular analysis by PCR (polymerase chain reaction). The PCR was performed for known SNPs of the BMP-15 gene (-9C&gt;G, 538G&gt;A, 788insTCT and 852C&gt;T) respectively. Amplified PCR products were sequenced commercially and analyzed against BMP-15 reference sequence using ClustalW2 application.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying MicroRNA-29a Improves Ovarian Function of Mice with Primary Ovarian Insufficiency by Targeting HMG-Box Transcription Factor/Wnt/β-Catenin Signaling.

Background Primary ovarian insufficiency (POI) is a female disease characterized by ovarian function loss under 40 years old. Transplantation of exosomes is an encouraging regenerative medicine method that has the potential for restoring ovarian functions post-POI with high efficiency. Therefore, we investigate the therapeutic efficacy and potential mechanisms of human umbilical cord mesenchymal stem cell- (UCMSC-) derived exosomes on ovarian dysfunction post-POI. Methods The model of POI was established by intraperitoneal injection with 5 mg/kg cisplatin. The mouse ovarian function was detected by measuring the levels of anti-Mullerian hormone, follicle-stimulating hormone, and estradiol and detecting the morphological changes. For in vitro experiments, the characterization and identification of UCMSCs and UCMSC-derived exosomes were done by observation of morphologies and flow cytometry. To exclude the interference effect of nonspecific precipitation substances, UCMSCs were treated with RNase A or RNase A in combination with Triton X-100. Granulosa cell (GC) identification was performed using immunofluorescence. GC proliferation and viability were assessed using 5-ethynyl-2′-deoxyuridine (EdU) assays and Cell Counting Kit-8 (CCK-8), and GC apoptosis was calculated by flow cytometry. Gene expression and protein levels were evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The binding relationship between miR-29a and HMG-box transcription factor (HBP1) was verified by luciferase reporter assays. Results In vitro, the human UCMSC-derived exosomes carrying miR-29a upregulation promoted the proliferation of GCs and suppressed their apoptosis. In vivo, miR-29a upregulation reserved the mature follicles and restored the ovarian functions. miR-29a targeted HBP1 and negatively regulated its expression. HBP1 upregulation rescued the miR-29a upregulation-induced inhibition in GC apoptosis and inactivated the Wnt/β-catenin pathway. Conclusion The exosomal miR-29a derived from human UCMSCs improves the ovarian function by targeting HBP1 and activating the Wnt/β-catenin pathway.

Vitamin D Supplementation Improves Follicle Maturation By Regulating Oxidant/Antioxidant Balance in Women With PCOS

Objective: To investigate the effect of vitamin D (VD) supplementation on follicular fluid total oxidant status (TOS) and total antioxidant status (TAS) in women undergoing IVF/ICSI due to PCOS. Materials and Methods: Forty infertile women who were diagnosed with PCOS and decided on IVF/ICSI and whose serum VD levels were lower than 20 ng/mL were included in the study. The VD levels of the patients were determined according to the proposal of Endocrine Society. The patients were divided into two equal groups with 20 patients in each group. While the patients in Group 1 were given 400 IU/day of oral VD3 replacement, the patients in Group 2 were given 600 IU/day of oral VD3. VD treatment was started one month before the controlled ovarian stimulation cycle and continued throughout the following cycle. Fifteen patients with serum VD levels &gt;30 ng/mL were taken as the control group. Oral VD replacement was not given to the patients in the control group. TAS and TOS values were measured in the follicle fluids taken from the patients in the VD replacement group and the patients in the control group on the day of ooist pick-up. Results: When compared with the control group, the follicular fluid TAS levels of both groups with VD replacement were significantly higher, while TOS levels were found to be significantly lower. When 400 IU/day VD replacement and 600 IU/day VD replacement were compared within themselves, follicular fluid TAS and TOS levels were found to be similar. Increasing the VD replacement dose from 400 IU/day to 600 IU/day did not cause a significant change in TAS and TOS values. A positive and significant correlation was found between intrafollicular TAS levels and serum VD, MII oocyte, 2PN zygote and clinical pregnancy rates. A negative correlation was found between intrafollicular TOS levels and serum VD, 2PN zygote and clinical pregnancy rates. Conclusions: VD replacement therapy contributes to follicle maturation by regulating intrafollicular oxidant/ antioxidant balance in PCOS patients.

The stimulation of ovulation and endometrial carcinogenesis: possible relationship and study prospects (literature review)

Currently, infertility is still a global problem; therefore, reproductive doctors worldwide are actively studying new and improving existing methods of overcoming female infertility. In recent years, the arsenal of drugs that can stimulate ovarian function has significantly expanded. A prerequisite for the use of drugs in this group is the presence of a follicular apparatus in the ovaries. Ovulation inducers contribute to the maturation of several follicles at once, and their ability to influence the process of steroidogenesis is beyond doubt. However, the long-term risk assessment when using these drugs, namely the risk of developing malignant female reproductive system neoplasm, remains poorly understood despite their long-term use. This problem is multifaceted and quite difficult to study since the etiological causes of infertility are considered a risk factor for developing malignant female reproductive system neoplasms. Oncological diseases are diagnosed several years after infertility treatment, thus proving a causal relationship is difficult, on the one hand, and dictating the need for a long period of observation in the group of women who underwent infertility treatment, considering the characteristics of each specific method, on the other hand. Herein, the authors analyzed the results of published studies on the possible relationship between ovulation induction and the risk of developing cancer in the uterine body.

P-788 A proof-of-concept study evaluating the effect of personalized dosages of follitropin delta in intra-uterine insemination (IUI): Personalized IUI Treatment Study (PITS)

Abstract Study question Would a personalized dosing regimen of follitropin delta based on AMH and body weight allow better management of women’s IUI cycle? Summary answer A personalized regimen of follitropin delta based on AMH and body weight has increased pregnancy rate without having increased multiple pregnancies in our studied population. What is known already IUI has been widely used as a common treatment for infertile couples due to its simplicity and low cost. Some studies have shown that cumulative live birth rate (LBR) can be increased by use of controlled ovarian stimulation. A recent meta-analysis reported that a protocol based on exogenous gonadotropins increases the LBR. Hence, this technique remains a treatment option reaching a LBR ranging from 8.5 to 12.2%. Nevertheless, this increase in LBR is associated with a non-negligible rate of adverse events, including ovarian hyper-stimulation syndrome (OHSS) and multiple pregnancies (7.2% - 9% of twins and 0.4% - 0.5% of triplets). Study design, size, duration This prospective, phase 2, open-label study enrolled 110 women 18 - 42 years of age from January 2020 to January 2021. Participants had received up to 3 IUIs. The primary efficacy variable was the number of cycles where target number of 2 or 3 mature follicles (MII) were reached. The secondary objective and safety variable were the incidence of cycle cancellations, the clinical pregnancy rate and the proportion of multiple pregnancies over 3 consecutive cycles. Participants/materials, setting, methods Follitropin delta dosing algorithm is modeled to achieve 8-14 oocytes in IVF cycles. To achieve 2-3 mature follicles, the algorithmically derived dose was divided by 4. Follitropin delta adjustment for subsequent IUIs were based on number of MII at previous IUI and age. Dose modification was made when excessive or no response were observed at stimulation day 8 during ultrasound monitoring. If urine pregnancy test was positive, a viability ultrasound was prescribed at 6-8 weeks. Main results and the role of chance Descriptive statistics were used in this proof-of-concept study. The mean age of all subjects was 34.46 years (± 4.5). The mean weight was 69.2 kg (± 11.24), AMH - 3.84 ng/ml (± 13), FSH - 6.3 IU (± 2.61), antral follicular count - 15 (± 8.6). The mean number of years of infertility was 2.48 ± 3.57. The percentage of patients who obtained 2 or 3 follicles during the 3 consecutive IUI was 34%, 37% and 47% respectively. The percentage of patients who reached ≥3 follicles was 1.9%, 0% and 1.4% respectively, demonstrating the ability of the proposed dosing regimen to achieve the desired goal. The percentage of cycle cancelation was stable throughout the 3 cycles: 8.5%, 2.4% and 8.8% respectively. Only 6 multiple pregnancies over 40 clinical pregnancies (15%) were observed, showing the proposed dosing regimen’s safe approach. The cumulative clinical pregnancy rate over the 3 cycles among patients completing the study was 37.7%, and the cumulative live birth rate (calculated ad hoc, as it was not part of the study protocol) was 33%, leading to the personalized dosing regimen proposed in this study to be considered for use in clinical practice. Limitations, reasons for caution The study’s limitations are its exploratory nature, open-label design and small sample size. Wider implications of the findings PITS study is the first one using follitropin delta for stimulation in intra-uterine insemination. It confirms the initial hypothesis of a potential benefit of a personalized dosing regimen of follitropin delta. A larger, randomized controlled study will be needed to confirm these initial findings. Trial registration number NCT03830723

P-467 Optimizing in vitro growth of immature follicles from cryopreserved vs fresh human cortical ovarian tissue

Abstract Study question Can cryopreserved-thawed human ovarian cortex be cultured in vitro to produce growing follicles as efficiently as from fresh samples? Summary answer In contrast to fresh samples, cryopreserved-thawed ovarian cortex did not develop morphologically normal growing follicles, even when adding follicular growth stimulators. What is known already A multistep culture system has been shown to produce mature oocytes from immature follicles present in the human cortex, without the addition of follicular growth stimulators. However, these promising results were achieved using fresh human ovarian cortex tissue donated from cisgender women, which is not available for the purpose of fertility preservation. It remains unclear whether this multistep culture system can be used to mature follicles from cryopreserved-thawed human ovarian cortex. In addition, the applicability of the culture system to mature follicles in vitro has not been investigated using ovarian tissue from transmasculine people. Study design, size, duration Fresh and cryopreserved ovarian cortex tissue isolated from the same transmasculine people were cultured during 8 days (first step of the culture system) with or without the addition of a PI3K/Akt promotor, Sphingosine-1-Phosphate (S1P). The ovarian cortex fragments were collected a day 0 and day 8 for downstream morphological and quantitative analysis of the follicular population. This study also included data of in vitro culture of cryopreserved samples from oncological cisgender women. Participants/materials, setting, methods Ovarian cortex tissue was collected from six testosterone user transmasculine people (25,8 ± 4,9 years) and three oncological cisgender women (24,0 ± 6,0 years). Fresh cortex was either directly cultured or previously cryopreserved. All ovarian fragments were cultured in the same conditions for 8 days. After the culture period, follicular population was quantified on histological sections (haematoxylin-eosin) and compared to samples of day 0. Follicular cell types, cell proliferation and apoptosis were assessed by immunofluorescence. Main results and the role of chance Fresh ovarian cortex tissue isolated from transmasculine people showed morphologically normal primary follicles (PFs) and secondary follicles (SFs) after 8 days of culture and no antral follicles were observed. Moreover, the addition of the follicular growth stimulator, S1P, to the culture medium significantly increased the number of growing follicles (PFs, P &amp;lt; 0.05; SFs, P &amp;lt; 0.01). The addition of S1P stimulated the emergence of SFs that showed expression of anti-mullerian hormone (AMH), contained proliferative (PCNA+) granulosa cells and showed low levels of apoptotic cells (TUNEL assay). Structurally, these in vitro grown SFs showed an intact basement membrane assessed by the expression of collagen IV. By contrast, using the same culture protocol but starting from cryopreserved-thawed cortex tissue from the same patients resulted in a very low number of viable growing follicles (PFs and SFs) even in the presence of S1P. Lastly, when using the same culture protocol on cryopreserved-thawed samples from oncological cisgender women, we observed a low efficiency of follicular growth, comparable to that observed in cryopreserved ovarian tissue from (age-matched) transmasculine people. Limitations, reasons for caution Limitations of this study are: small sample size and lack of fresh cortex tissue from oncological cisgender women. Due to the high variability of follicular density among patients, the use of a larger number of samples is recommended. Wider implications of the findings Optimization of culture systems to grow and mature follicles in the ovarian cortex of cryopreserved samples (both from cis and trans persons) is urgently needed. Current culture protocols can be applied to fresh samples, but further characterization is needed to consider this as an alternative in fertility preservation treatment. Trial registration number Not applicable

P-657 Administration of an extra Gonadotropin dose on trigger day in patients undergoing elective egg freezing

Abstract Study question Does an extra Gonadotropin (GT) dose on trigger day during a gonadotropin-releasing hormone (GnRH) antagonist cycle for oocyte cryopreservation result in improved cycle outcomes? Summary answer An extra GT dose on trigger day in patients undergoing elective egg freezing is not associated with increased oocyte yield. What is known already The most common controlled ovarian hyperstimulation (COHS) protocol used in elective oocyte cryopreservation cycles is a GnRH antagonist protocol with a GnRH agonist trigger. There is no consensus about the timing of the final gonadotropin dose administration. Some physicians add an extra GT dose during the ovulation trigger day and others advise to receive the last GT dose the day before. The yield of this extra GT dose was not previously evaluated. Study design, size, duration A retrospective cohort study, consisting of cycles of elective oocyte cryopreservation at Hadassah Medical Center from 1.2017 through 5.2021. Patients were divided into 2 groups- women who received their last GT dose a day before the GnRH agonist trigger, and women who received the last GT on the trigger day. Background and gynecologic characteristics, as well as COHS cycle parameters, were compared. The primary outcome was the average number of mature oocytes in each group. Participants/materials, setting, methods Women 30-41 years who were admitted for elective oocyte cryopreservation. Women who were referred due to medical reasons or cycles with a protocol other than GnRH antagonist were excluded. Ovarian stimulation with GT began on the 3rd day of the cycle, and five days later, a GnRH antagonist was added. When more than two mature follicles were demonstrated, a GnRH agonist trigger was administrated and approximately 36 hours later, the oocyte aspiration procedure was performed Main results and the role of chance During the study period, 448 elective egg freezing cycles were performed, 151 cycles with an extra dose on the trigger day and 297 cycles without an extra GT dose. The groups did not differ in their background characteristics, age, BMI, GT used, and the number of induction days. However, the extra GT group had significantly higher day 3 follicular stimulating hormone (FSH), lower anti-Mullerian hormone (AMH), and lower maximal Estradiol (E2) levels. The extra GT dose group had a significantly lower mean number of mature oocytes (11.1±7.1 vs. 6.4±4.5, p &amp;lt; 0.001). To adjust for the maximal E2 parameter, that differed between the groups, an additional analysis was performed, dividing the two groups into subgroups of cycles with maximal E2 above and below 10,000 pmol\l. Interestingly, even in cycles with maximal E2 above 10,000 pmol\l, the extra dose group had poorer results (10.3±4.3 vs. 12.7±7.2, p &amp;lt; 0.001). This difference was also noted in cycles with E2 below 10,000 pmol\l (5.4±4.0 vs 8.7±6.1 mature oocytes, p = 0.011). Additionally, a multiple regression model for the prediction of several oocytes retrieved showed that the extra GT dose parameter was not associated with increased oocytes number. Limitations, reasons for caution The retrospective design of this study and the differences in cycle characteristics between patients that did or did not receive an extra GT dose on trigger day make it prone to selection bias. Wider implications of the findings Our study shows that administration of an extra GT dose on trigger day in oocyte cryopreservation cycles does not yields more mature oocytes. However, wider prospective studies are needed to thoroughly investigate the implications of this treatment’s effect on cycle outcomes. Trial registration number Not applicable

P-574 Ovarian Revitalisation using Platelet Rich Plasma

Abstract Study question Can we rejuvinate ovaries (with low ovarian reserve) by injecting fresh platelet rich plasma ? Summary answer With ovarian PRP injections, Ovarian reserve will increase and number of follicles increases in treated ovaries . What is known already Till date no definitive treatment is available to restore function of ovaries. Few drugs e.g. granulocyte colony stimulating factor etc tried for the same but no satisfactory result was achieved. Oral ovulogens and gonadotropins cannot produce satisfactory number of ova in these patients. So they were forced to go for donar ova. By using ovarian PRP injections couple can have a chance of their own biological child. Study design, size, duration we conducted the pilot study involving injection of autologous fresh platelet rich plasma injection into the ovaries of subfertile females by comparison of their fertility parameters viz. hormonal profile involving measurements of blood levels of follicular stimulating hormone (FSH), luteinizing hormone (LH), anti-Mullerian hormone (AMH), prostaglandin E2 levels (PGE2); antral follicular count (AFC); number of mature follicles (NMF) and number of retrieved oocytes (NRO) in both pre-PRP and post-PRP states. Participants/materials, setting, methods Forty-five subfertile females of more than 20 years were included in our study . All the patients underwent fresh autologous PRP injection under transvaginal ultrasonographic guidance. Besides the demographic data, each patient’s hormonal profile (including blood levels of AMH, FSH, LH, PGE2), AFC, NMF and NRO in both pre-PRP and post-PRP state were recorded in a predesigned proforma. Statistical evaluation of the recorded data was done using appropriate methods and tools. Main results and the role of chance Mean age, weight, height, body mass index and years of subfertility of the patients in our study was 31.27yrs, 58.38kg, 154.84cm, 24.37kg/m2 and 8.44years respectively. The mean blood levels of AMH increased significantly in post-PRP state (1.6 vs 2.4pg/ml) in contrast to significant decrease in blood level of FSH (21.4 vs 12.5mIU/ml), LH (15.1 vs 5.9mIU/ml) &amp; PGE2 (162.7 vs 66pg/ml). The AFC, NMF &amp; NRO also increased in post-PRP state relative to pre-PRP state. The differences in above levels and numbers were statistically significant. Limitations, reasons for caution Autologous PRP injection into ovaries improves hormonal profile, AFC and number of mature follicles as well as retrieved oocytes. Furthur followup is needed to know weather our patients concieve spontaneously or they will need some kind of assisted tecniques to concieve a pregnency. Wider implications of the findings Autologous PRP injection into ovaries improves hormonal profile, AFC and number of mature follicles as well as retrieved oocytes, it can be utilized as a revitalisation tool to boost fertility. Our study results reinforce the above concept. However, outcome-based studies are needed to further validate the results of our study. Trial registration number TMU/IEC/19-20/0113

P-362 The association between pregnancy-test day serum progesterone in IVF pregnancies and obstetrical complications

Abstract Study question Is there an association between pregnancy-test day serum progesterone level in IVF pregnancies and late gestational complications? Summary answer Low serum progesterone on pregnancy diagnosis day is independently associated with a higher prevalence of preeclampsia. What is known already Low first trimester arbitrary serum progesterone is associated with early pregnancy complications such as ectopic pregnancies and miscarriages. IVF pregnancies are associated with higher prevalence of obstetrical complications such as preterm birth, preeclampsia and small for gestational age. Different patient and treatment parameters such as age, diagnosis, peak blood Estradiol level, endometrial thickness, fresh or frozen embryo transfer are associated with obstetrical complications. Progesterone is a potent immunomodulator that can affect the inflammatory pathway and trophoblast invasion. Little is known about the association between early pregnancy progesterone level and obstetrical complications, with conflicting results. Study design, size, duration A cohort study reviewing fertility and delivery files of all singleton live births from successfully treated infertile patients, who underwent ovarian stimulation, IVF and fresh embryo transfer in a tertiary medical center between 2008 and 2018. Participants/materials, setting, methods Serum progesterone concentration was measured with the first serum hCG on 4 + 0 gestational week (14 days after oocyte retrieval) under luteal support with vaginal progesterone. Pregnancy-test day serum progesterone was categorized in quartiles. The prevalence of obstetrical complications including gestational diabetes mellitus, preterm birth, preeclamsia, small for gestational age, post-partum hemorrhage and neonatal ICU hospitalization was compared between the four groups. Confounders were adjusted for by multivariate analysis. Main results and the role of chance 719 singleton live births were included. The four serum progesterone quartiles were: Q1:3-44 nmol/L(n = 180); Q2:45-107 nmol/L(n = 180); Q3:108-223 nmol/L(n = 178); Q4:225-808nmol/L(n = 181). Patients with lower progesterone levels (Q1) had significantly more IVF treatments, lower peak estradiol level, fewer mature follicles and frozen embryos. The incidence of preterm labor, gestational diabetes mellitus, small-for-gestational-age, post-partum hemorrhage, placental abruption or neonatal ICU administration did not differ between the groups. Patients with lower serum progesterone had higher incidence of preeclampsia (9.44% in Q1, 2.78% in Q2, 2.81% in Q3 and 3.31% in Q4, P = 0.0046). On multivariate analysis, controlling for age, gravidity, treatment number, number of oocytes, peak follicular estradiol and progesterone, Q1 progesterone level was independently associated with an increased risk of preeclampsia (estimated OR = 3.27 95% CI 1.455-7.299). Limitations, reasons for caution Retrospective design, vaginal progesterone luteal support administration. Wider implications of the findings Low serum progesterone on pregnancy diagnosis day in gestations resulting from fresh ET is associated with a significantly higher prevalence of preeclampsia, possibly because of aberrant placentation and vascular development. This association is important for the obstetrical management, preeclampsia risk assessment, and administration of preventive therapy. Trial registration number not applicable

P-695 Anti-muellerian Hormone (AMH) and Plasminogen Activator Inhibitor (PAI-1) are robust follicular-fluid (FF) biomarkers predictive of follicle maturity and oocyte quality in women undergoing IVF cycles

Abstract Study question Is AMH the in-vivo trigger that correlates with PAI-1 in the follicular-fluid micro-milieu to influence maturity and quality of oocytes derived in vitro during IVF? Summary answer Lower FF-AMH levels correlate with top quality oocytes whereas Lower FF-PAI-1 levels correlate with follicle-maturity as reflected in mature MII oocytes retrieved in IVF cycles What is known already Ovulation is a time-coordinated tightly regulated proteolytic event. PAI-1, primarily produced by granulosa cells, is a major form of PAI in FF of human preovulatory follicles, allowing localized degradation and regulating ovulation.While ovulation process is rendered redundant in IVF cycles; nevertheless FF PAI-1 concentration may influence maturity of oocyte within the follicle. AMH is exclusively produced by granulosa cells of pre-antral &amp; early-antral ovarian follicles in the adult female and secreted into FF. Although AMH is widely accepted as an ovarian-reserve marker, very few studies have explored direct putative correlation of FF-AMH with oocyte quality in stimulated IVF cycles. Study design, size, duration Prospective study of n = 1150 (Power of study &amp;gt;85%) women (mean age 30.22±4.25 years, BMI 23.97±4.53, W/H ratio 0.88±0.06) undergoing antagonist stimulation protocol with r-FSH (September 2015-December 2018). On day of ovum pick-up, original aspirate per follicle was pooled per patient. AMH and PAI-1 were evaluated in pooled FF by ELISA method using diagnostic kits. These protein-biomolecules were expressed as ratio of total protein content. SerumE2, LH, P on d/hCG trigger were also estimated by RadioImmunoAssay. Participants/materials, setting, methods Elderly women (age&amp;gt;35 years), women with polycystic-ovaries, endometriosis were excluded. Embryo-transfer (ET) was done either on Day3 or day5. Luteal-phase was supported with micronized progesterone. All cycles were divided into Low FF-AMH (≤1.72 ng/mg protein), High FF-AMH (&amp;gt;1.72ng/mg protein) and Low FFPAI-1 (≤592.1 ng/mg protein), High FFAI-1 (&amp;gt;592.1 ng/mg protein) groups. Oocyte quality was graded as poor, average and top based on morphological assessment of COC-complex, zona-pellucida (thickness, regularity) and cytoplasm (granularity, vacuolation, refractile bodies). Main results and the role of chance Mean Age, Baseline D3-Serum AMH, E2 d/hCG and number of eggs retrieved did not differ significantly between low vs. High FF AMH groups as well as Low vs. High FF-PAI-1 groups. Low FF AMH group displayed significantly higher percent of top-quality oocytes (70.38±11.5 vs. 52.16±8.3;p&amp;lt;0.0001) and higher fertilization rates (88.68±10.7 vs. 72.5±9.2%; p = 0.004) compared to High FF AMH group. Incidences of total fertilization failure were significantly higher in high FF AMH (3.25% vs. 1.1%, p = 0.0002) compared to Low-FFAMH group. FF-AMH correlated with oocyte-quality (Pearsonr=0.48); lower-levels increased the odds of top-quality oocytes. Thresholds of FF-AMH for top quality oocytes was &amp;lt;1.8 ng/mg protein;ROCAUC:85%) Low FF PAI-1 group had significantly higher Percentage of MII oocytes (93.55 vs. 74.8%; p &amp;lt; 0.0001), Rates of Fertilization (86.35 vs. 71.04, p = 0.008) and cleavage (79.19 vs. 69.85;p=0.03) compared to High FF-PAI-1 group. FF-PAI-1 correlated with oocyte-maturity (Pearsonr=0.51), lower-levels increased the odds of obtaining MII-oocytes. Cutoff of FF PAI-1 for mature MII oocytes was &amp;lt;504.4 ng/mg protein;ROCAUC:76%). FF-AMH showed strong positive correlation with FF-PAI-1 (Pearsonr=0.66) Limitations, reasons for caution Several other molecules in the follicular-fluid milieu may be responsible for maturity status and quality of oocytes. Although these are apparently the most plausible of markers; role of other factors cannot be ruled out. Also, it may be better to study mono-follicular aspirates rather than pooled FF for each patient. Wider implications of the findings FF-levels of AMH and PAI-1 as non-invasive endogenous biomarkers of oocyte maturity and quality in-vitro may facilitate better understanding of the physiological pathway governing in-vivo ovulation process. This study reiterates ‘Follicle-maturation’ is the rate-determining step that sets off a cascade mechanism triggering oocyte-maturation and quality further influencing fertilization, embryo development. Trial registration number In patients with polycystic ovarian syndrome (PCOS), are serum androgen levels (total and free testosterone) associated with the risk of endometrial hyperplasia or cancer?

Comparing Naltrexone and Metformin Pretreatment for Inducing Ovulation in Patients With Polycystic Ovary Syndrome in Intrauterine Insemination Cycles

Objectives: Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women. Hyperinsulinemia and insulin resistance are normally observed in PCOS patients and metformin is used to treat this disease. The evidence indicates that the opioid system plays a role in the pathogenesis of the PCOS. Based on the above-mentioned explanation, the present study evaluated the role of opioid antagonist (naltrexone) in PCOS-induced infertility compared to metformin. Materials and Methods: Totally, 120 patients afflicted with PCOS were assigned to three groups based on Rotterdam diagnostic criteria, with a body mass index (BMI) of 25-30 kg/m2 . The first group received naltrexone (50 mg/d) for 8 weeks. In addition, the second group were injected with metformin 1000 and then 1500 mg/d for the first and seventh weeks, respectively. Finally, the third group entered the intrauterine insemination (IUI) without prior therapeutic treatment. Then, the groups were compared in terms of the amount of estradiol produced per day of human chorionic gonadotropin injection and the total amount of gonadotropin needed, the number and size of the adult follicles, the number of days of taking the drug, and incidence of pregnancy-induced abo Results: Based on the results, the mean of infertility duration was not significant among the three groups (P=0.782). Further, the mean fasting insulin level between the metformin and control groups represented a significant difference (P=0.045). The average number of days to trigger patients in the 3 groups was not significant (P=0.346). Although the average number of follicles between metformin and naltrexone groups was not significant, it was higher in the naltrexone group. Finally, the average BMI of the naltrexone group after the treatment was lower compared to before treatment (P≤ 0.001). Conclusions: In general, the number of receiving days and the dose of the drug in the naltrexone group was lower compared to the metformin group. Furthermore, the number of mature follicles in both ovaries and the serum estradiol level in the naltrexone group was higher compared to the metformin group.

Protective Effect of XinJiaCongRongTuSiZiWan on the Reproductive Toxicity of Female Rats Induced by Triptolide.

Background Although triptolide (TP) has been widely used for the treatment of inflammatory, autoimmune diseases, and various kinds of tumors, the long experimental and clinical applications have exhibited severe reproductive system toxicity in TP-treated animals and patients. More importantly, the underlying molecular mechanism involved in TP-induced reproductive system toxicity still needs more research. Methods Adult female Sprague Dawley rats and human ovarian granulosa cell lines were treated with TP and then treated with XinJiaCongRongTuSiZiWan (XJCRTSZW). Histological analysis and follicle count were executed using H&E staining. Hormone (E2, AMH, FSH, LH, and INH B) concentrations, inflammation indicators (IL-1β, IL-6, and TNF-α), oxidative stress indicators (SOD, GSH-Px, and MDA), apoptosis rate, protein distribution and expression (SIRT1, AMPK, and 8-OhdG), cell viability, relative protein levels (beclin-1, LC3-II/LC3-I, p62, procaspase-3, cleaved caspase-3, p-SIRT1, SIRT1, p-AMPKα-1, AMPKα-1, Akt, and p-Akt), autophagosome were detected by ELISA, commercial biochemical detection kits, flow cytometry, immunohistochemistry, CCK-8, western blotting, and transmission electron microscope, respectively. Results XJCRTSZW administration notably improved the TP-treated pathological symptoms, including few mature follicles in the ovary and less granular cell layer, and disordered the arrangement of the follicle, lymphocytes and plasma cells infiltration, and necrosis, shedding, and follicular cystic dilatation of the granular layer follicle cells in the ovarian stroma. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of primary follicles and secondary follicles numbers and decreased the TP-induced elevation of atretic follicle numbers and the expression of AMPK, SIRT1, and 8-OhdG in GCs in vivo. Moreover, XJCRTSZW application significantly increased the TP-induced diminishment of E2, AMH, and LNH-B concentrations, apoptosis rate, SOD and GSH-Px concentrations, and p62 protein level; however, it declined the TP-induced augmentation of MDA level, the levels of IL-1β, IL-6, and TNF-α, autophagosome, beclin-1, LC3-II/LC3-I, cleaved-caspase-3, p-AMPKα-1, and p-SIRT1 protein levels both in vivo and in vitro. Besides, XJCRTSZW treatment prominently enhanced the TP-induced decrease of cell viability in vitro. Conclusion XJCRTSZW can alleviate TP-induced reproductive toxicity via apoptosis, inflammation, and oxidative stress both in vivo and in vitro. Moreover, XJCRTSZW ameliorates TP-induced reproductive toxicity through AMPK/SIRT and Akt signaling axis mediated autophagy both in vivo and in vitro.