Ovarian Puncture Research Articles

Ovarian Puncture Triggers an Inflammatory Response that did not Affect Late Folliculogenesis, Ovulation Rate, and Fertility.

Ovarian puncture has been widely used in assisted reproduction, but there are still gaps about its effects on ovarian morphophysiology, as well as the relationship between inflammation caused by this procedure and the follicular growth and fertility. The aim of this study was to investigate the effects of ovarian puncture on folliculogenesis and fertility. Mice (n = 24) were divided into two groups: (1) SHAM-both ovaries were exposed and repositioned and (2) Punctured-ovaries were exposed, punctured, and repositioned. After 96h of surgery, ovaries were collected for morphofunctional analysis. New females were used for the superovulation (n = 10) and fertility assays (n = 10). Increased volumetric density of inflammatory cells-p = 0.0005, p = 0.0013; hemorrhagic foci-p < 0.0001; and inflammatory exudate-p < 0.0001 could be noticed on the punctured group, compared to SHAM. The percentage of primordial follicles was lower on the punctured ovaries (p = 0.00294). Ovarian puncture has also induced an increase in the proliferation of granulosa cells of primary (p = 0.0321) and antral follicles (p = 0.0395), and an increased apoptotic index of antral follicles (p = 0.0100). There was no influence on expression of some genes related to inflammation, collagen deposition and folliculogenesis progression. The reproductive aspects (oocyte retrieval and number of fetuses per female) were not altered (p > 0.05). Taken together, our findings strongly suggest that ovarian puncture results in a local inflammation that affects follicular growth and atresia. However, it does not affect female fertility, which strengthens the safety of this procedure.

Oocyte Competence, Embryological Outcomes and miRNA Signature of Different Sized Follicles from Poor Responder Patients

Poor ovarian response (POR) patients often face the risk of not having enough competent oocytes. Then, aspirating small follicles could serve as a strategy to increase their number. Many efforts have been addressed to associate follicular size with oocyte competence, but results are controversial. Therefore, our study aimed to evaluate oocyte maturation and developmental competence, along with a non-invasive oocyte-maturation-related miRNA signature in oocytes retrieved from both large and small follicles. A total of 178 follicles, from 31 POR patients, were aspirated and measured on the day of ovarian puncture. Follicular diameters, oocyte collection, oocyte maturation, fertilization, blastocysts, and good-quality blastocyst rates were recorded. Simultaneously, follicular fluids were collected to quantify their miRNA expression. The efficacy of oocyte retrieval along with oocyte maturation, fertilization, and blastulation rates tended to increase with follicular size, but few significant differences were found. Despite there being significantly more collected oocytes from follicles &gt; 11.5 mm compared to follicles ≤ 11.5 mm (p &lt; 0.05), oocytes from the latter were also mature, with no significant differences in the miRNA signature, but only those &gt; 13.5 mm demonstrated developmental competence. In conclusion, 11.5 mm follicles can produce mature oocytes, but only those larger than 13.5 mm yielded transferable embryos.

Blood type ABO and the cytokine profile of follicular fluid in women undergoing IVF/ET.

ABO blood type was hypothesised to be related to a number of infertility processes. There is still an open debate on ABO blood group's incompatibility and infertility. It was associated with ovarian reserve in women with subfertility. There is still not enough information on the influence of blood type and the immunology of follicular fluid (FF). 78 patients were selected, who underwent in vitro fertilization (IVF) between April 2021 and January 2022. FF samples from each individual patient were taken on the day of ovarian puncture and stored at -80°C until immunological assessment. Concentration of chosen interleukins - IL-1α, IL-2, IL-4, IL-5, IL-6, IL-8 IL-10, IL-15, IL-1β, IL-18, IFN, LIF, TNFα, GCSF and PIBF-1 were measured using commercially available ELISA kits. All assessed cytokines were present in the FF of exanimated patients. The concentration was compared to the blood type ABO of all women undergoing in vitro fertilization. No statistical relevance was found between blood type ABO and the concentration of GCSF, PIBF1, LIF, IL-15, IL-5, IL-8, IL-1 alfa, IL-1 beta, INF gamma, IL-2HS, IL-4HS, IL-6HS, IL-10HS in the FF obtained during ovarian puncture (p > 0,05). There was no statistically significant correlation between blood type ABO and the quality of embryo, and the positive pregnancy test in patients undergoing IVF/ET. The blood type ABO does not influence the wide cytokine profile of FF obtained during ovarian puncture in women with infertility of different origin, as well as embryo quality and pregnancy rate.

P-629 In vivo follicular activation (IVFA) through repeated ovarian punction: results of an experimental study in a premature ovarian insufficiency rodent model

Abstract Study question Can the new IVFA therapy effectively promote ovarian follicle development and increase oocyte number in a premature ovarian insufficiency (POI) animal model. Summary answer The IVFA technique reveals favourable reproductive outcomes in the animal model, demonstrating increased oocyte retrieval without compromising quality, making a promising advancement in fertility restoration. What is known already POI is characterized by the loss of the primordial follicular pool before the age of 40. Ovarian fragmentation for in vitro follicular activation (IVA) is a technique based on the disruption of the ovarian extracellular matrix, subsequently promoting actin polymerization and interfering with the evolutionarily conserved tumour suppressor Salvador-Hippo-Warts (SHW) signalling pathway. This has been proven to increase expression of downstream growth factors, thus promoting the primordial follicle growth and the generation of mature oocytes. Yet, this procedure is invasive and expensive. Consequently, a demand exists for less invasive in vivo therapies. Study design, size, duration Experimental animal study. Sixty 6-week-old C57BL/6 female mice were divided into control (CT) and POI groups. Each group, was further divided into three subsets (n = 10/group): without ovarian punctures (CT-0 and POI-0), 15 punctures (CT-15 and POI-15), and 22 punctures (CT-22 and POI-22). Twenty-one days after POI induction, the new IVFA technique was performed on the anaesthetized animals. Subsequently, controlled ovarian stimulation (COS) was initiated, followed by euthanasia for the oocyte retrieval and histological ovarian evaluation. Participants/materials, setting, methods POI condition was induced with intraperitoneally injections of 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan. The IVFA technique involved ovarian punctures using a 30-gauge needle. COS was induced with 10 IU of PMSG and 10 IU of hCG 48 hours later, leading to euthanasia for oocytes and ovaries collection. Ovaries (n = 6/group) were fixed for follicular counts and immunohistochemistry. Oocyte quality assessment included reactive oxygen species (ROS) levels, mitochondrial fluorescence (Mitotracker), and spindle analysis. Main results and the role of chance The control groups subjected to the IVFA technique (CT-15 and CT-22) demonstrated significantly higher mean total oocyte counts compared to the non-intervention group (CT-0) (30.0±9.8 and 27.7±7.9 vs 18.1±11.7, respectively; p-value&amp;lt;0.05 in both comparisons). Similarly, within the POI model, interventions (POI-15 and POI-22) showed significantly elevated mean total oocyte counts than the non-intervention group (POI-0) (8.1±4.4 and 7.2±2.8 vs 3.8±3.6, respectively; p-value&amp;lt;0.05). The intensity of ROS fluorescence, Mitotracker fluorescence, and spindle analysis in retrieved oocytes were not affected by the IVFA therapy, thereby preserving oocyte quality. Notably, the percentage of growing follicles was significantly increased in the intervention groups compared to the non-intervention groups in the control (60±5.7% and 58.1±4.7% vs 41.5±4.5%; p-value&amp;lt;0.001). In the POI model, statistically significant differences were observed for POI-15 vs POI-0 (57.8±9% vs 41.6±13.3%; p-value&amp;lt;0.05), but not for POI-22 vs POI-0 (51.5±14.2% vs 41.6±13.3%, p=n.s.) Moreover, the global Ki67 proliferation marker demonstrated a significant increase in the percentage of proliferative cells in the CT-15 intervention group compared to the CT-0 group (53±11.2% vs 46.1±15.1; p &amp;lt; 0.01). Finally, in the POI model, the percentage of Ki-67 positive cells was also significantly higher in both IVFA groups (39.4±18.5% and 38.1±17.3% vs 32±17.5%, respectively; p &amp;lt; 0.05). Limitations, reasons for caution Since this is an animal model of POI, further validation of the new therapy with human ovarian samples would be required. The magnitude of the effect could vary due to inter-species variability. Wider implications of the findings The positive results of the new therapy in promoting follicle development and increasing oocyte retrieval, without compromising quality, have significant implications for developing new fertility treatments. If validated in humans, this approach could offer a promising solution for women with POI. Trial registration number not applicable

P-369 DuoStim could be a proper option for women seeking fertility preservation for non-medical reasons

Abstract Study question Is Duostim a suitable option for women who wish to preserve fertility for social reasons? Summary answer Providing the option of DuoStim is an efficient way to expand the cohort of vitrified oocytes in women seeking fertility preservation for non-medical reasons. What is known already A growing number of reproductive-age women are choosing to postpone motherhood, giving rise to the concept of reproductive autonomy. Numerous strategies aim to refine ovarian stimulation protocols. DuoStim, entailing dual ovarian stimulations and retrievals within one menstrual cycle, proves advantageous for those with diminished ovarian reserve or facing cancer. To our knowledge, this is the first study offering DuoStim for social preservation. Study design, size, duration Retrospective controlled case-control study conducted with paired follicular phase stimulation (FPS) and luteal phase stimulation (LPS) derived cohorts of oocytes collected in the same ovarian cycle (DuoStim). The study included 160 patients aged 36.85 ± 3.3 years old, who underwent DuoStim for social egg freezing from October 2018 to September 2023. The primary outcome was the number of MII oocytes retrieved per cycle and secondary outcomes included stimulation duration and gonadotropin dose. Participants/materials, setting, methods FPS and LPS were performed using the same daily dose of recombinant gonadotropins in an antagonist protocol. Oocyte retrieval was performed 36 hours after triggering with gonadotropin-releasing hormone agonist triptorelin acetate (0,2 mg) after the FPS. Luteal phase stimulation protocol began on the 5th day after ovarian puncture using dual trigger, followed by a second oocyte retrieval. Mature oocytes were vitrified 2 hours later following the Kitazato® protocol. Main results and the role of chance Stimulation duration and gonadotropin dose were lower during Follicular Phase Stimulation (FPS) vs. Luteal Phase Stimulation (LPS) (10.8 ± 1.5 days vs. 13.0 ± 2.3, P &amp;lt; 0.05 and 2,487.9 ± 580.8 vs. 3,264.0 ± 942.7, P &amp;lt; 0.05). The number of retrieved oocytes was comparable between the two groups (4.78 ± 3.0 vs. 5.32 ± 3.7; P = 0.15). The mean number of MII oocytes acquired in the LPS group exceeded that in the FPS group (4.16 ± 3.0 vs. 3.58 ± 2.3; P = 0.054), indicating a tendency towards more MII oocytes in the LPS group without achieving statistical significance. A total of 110 patients (69.29%) obtained the same or a greater number of MII during the LPS compared to the FPS. Limitations, reasons for caution Although DuoStim is acknowledged as an option to obtain a greater number of eggs in certain situations, the evidence supporting its utility in social fertility preservation is still limited. It is crucial to recognize the inherent biases in observational retrospective studies. Wider implications of the findings DuoStim could be an advantage for women who live abroad or those who dispose of short periods of time for oocyte vitrification, also potentially reducing the drop-out rate. Trial registration number not applicable

Endometrial biopsy performed before the first in vitro fertilization does not impact the early pregnancy rate

Endometrial biopsy (EB) has been showed to increase the rate of clinical pregnancy in patients who underwent in vitro fertilization (IVF) failures. The purpose of this work was to assess the impact of an EB performed before the first in IVF on the early pregnancy rate. Be One study is a prospective, single-centre, randomized, open-label study. In this parallel study, patients were evenly split into two groups. In one group, patients underwent an EB between days 17 and 22 of the menstrual cycle that precedes the ovarian stimulation. In the other group (control), no EB was performed. The hCG-positive rate (early pregnancy rate) was evaluated on day 14 after the ovarian puncture. In total, 157 patients were randomized in the EB group and 154 patients were in the control group. The early pregnancy rate was 33.1% (52/157) in the EB group and 29.9% (46/154) in the control group (p = 0.54). Other parameters, including perforation, endometritis, or pain level were reassuring. An EB performed during the luteal phase of the menstrual cycle preceding the stimulation of the first IVF did not increase early pregnancy rate.

Comparison of Follicular Fluid Glycosaminoglycan and Hydroxyproline Concentration in Women with Polycystic Ovary Syndrome with Healthy Women

Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies in women of childbearing age. As a complex structure, the extracellular matrix (ECM) and its components, including glycosaminoglycans (GAGs) and collagens, are highly affected by hormonal alterations. Objectives: This study aimed to evaluate the level of GAG and hydroxyproline in the follicular fluid (FF) of women with PCOS. Methods: Overall, 62 women referring to Fatemieh Hospital Infertility Center in Hamadan were evaluated in this study. FF samples were taken from 31 women with PCOS during ovarian puncture and 31 healthy women. Oocytes were evaluated in the different stages of maturation, namely, metaphase II (MII) and germinal vesicle (GV). Commercially available kits were used to measure the levels of glycosaminoglycans and hydroxyproline. Results: The results of the present study showed that the count of oocytes in women with PCOS was significantly higher than that in healthy women. Evaluating GAG and hydroxyproline levels indicated that their levels were meaningfully higher in the FF of women with PCOS. Conclusion: In general, the results of this study suggested that ECM changes in PCOS followed by alterations in the levels of GAGs and hydroxyproline may lead to an increase in the level of GAG and hydroxyproline in the FF. The evaluation of these markers may be the subject of further studies.

The Role of Oxidative Stress in Infertility.

(1) Background: Oxidative stress markers in the follicular fluid and serum of the patient with IVF results (number of fertilized oocytes, number of embryos, cumulative pregnancy rate) are important in establishing the causes of infertility. (2) Methods: 42 patients were enrolled in the study over the duration of 24 months and were divided into two groups: the study group divided into the female etiology for infertility as a tubal factor, diminished ovarian reserve, endometriosis, and unexplained infertility, and the control group consisting of the male factor, excluding the sperm donors and with no female factor cause. On the day of ovarian puncture in IVF, follicular fluid and serum were collected from the patients. (3) Results: Malondialdehyde from the follicular fluid was higher in the control group. The total antioxidant capacity in the follicular fluid is positively correlated with the pregnancy rate. There is no statistically significant difference in the oxidative stress markers from serum in both groups. (4) Conclusions: The capacity of the follicular fluid environment to contain the reactive oxygen species (ROS) leads to a higher pregnancy rate in the control group.

P-673 Women exposed to higher levels of DEHP display altered microRNA expression profile in the pre-ovulatory follicular fluid

Abstract Study question Can higher concentrations of di-2-ethylhexyl phthalate (DEHP) in follicular fluid (FF) lead to an altered microRNA expression profile? Summary answer The present study indicates the upregulation of miR-203a-3p, miR-150-5p and miR-28-3p in the follicular fluid of women with higher DEHP concentrations (FDR&amp;lt;0.05). What is known already Women are exposed daily to endocrine-disrupting chemicals (EDCs) such as phthalates which can be found in hundreds of personal care products and plastic items. In studies of rodents exposed to certain phthalates, high doses have been shown to change hormone levels, disrupt folliculogenesis and cause birth defects. Recently, we reported a strong inverse association between DEHP metabolites and ovarian sensitivity index (OSI) in females undergoing ART treatment. Several mechanisms have been proposed to explain the association between DEHP and fertility in rodents, however not much is known about the mechanisms in humans. Study design, size, duration FF was collected from 96 women undergoing IVF treatment after controlled ovarian stimulation during ovarian puncture. 48 participants from Estonia were recruited at Nova Vita Clinic AS in Tallinn, Estonia in 2019 and 48 participants from Sweden were recruited at Carl von Linnekliniken in Uppsala, Sweden in 2016. In both cohorts, patients signed a written informed consent form. Both cohorts were matched for age, BMI, parity and the number of previous IVF treatments. Participants/materials, setting, methods DEHP metabolite concentrations in the FF samples were measured in our earlier study by LC-MS/MS. Cell free RNA was extracted from FF samples with Qiagen miRNeasy Micro kit, sequencing libraries were prepared with QIAseq miRNA library kit and sequenced on Illumina NextSeq 550 instrument. Differential miRNA expression analysis was performed with the DESeq2 package. Pathway enrichment analysis for differentially expressed miRNA targets was conducted with the miEAA tool integrated with WikiPathways. Main results and the role of chance In total, 465 miRNAs were observed at least twice in ≥ 24 samples. Differential expression analysis was conducted between DEHP-high and DEHP-low groups in combined cohorts using the cohort as a covariate. 16 miRNAs were found to be differentially expressed between DEHP groups with FDR&amp;lt;0.1. Three miRNAs: miR-203a-3p, miR-150-5p and miR-28-3p, were differentially expressed with FDR&amp;lt;0.05 and were more abundant in women with high DEHP-levels in both cohorts. Of these, miR-203a-3p was negatively correlated with OSI (Pearson R = -0.27, p = 0.0089). Six miRNAs (miR-10b-5p, miR-112-5p, miR-132-5p, miR-203-3p, miR-205-5p and miR-27-3p) were found to have predicted targets in the androgen receptor signalling pathway. Limitations, reasons for caution The miRNAs found in this study need to be validated in a larger set of patients as a cohort-dependent difference in the results was observed. There is a need for further functional studies to investigate miRNAs as possible mediators of the association between DEHP and ovarian sensitivity index. Wider implications of the findings The current study presents evidence that DEHP exposure alters the expression of FF miRNAs which are associated with androgen receptor signalling. These results provide novel evidence of the potential influence of endocrine-disrupting chemicals on female reproduction. Trial registration number NA

Эффективность применения различных форм гестагенов для поддержки лютеиновой фазы в протоколах ЭКО. Ретроспективное исследование

Objective. To compare the efficacy of different forms of gestagen administration for luteal phase support in in vitro fertilization (IVF) protocols with gonadotropin-releasing hormone antagonists. Patients and methods. Data on ART cycles with “fresh” embryo transfers (day 5) were collected in a retrospective study. A total of 154 patients were divided into three groups. Group 1 (n = 100) received oral dydrogesterone 10 mg 3 times daily as luteal phase support (LPS), group 2 (n = 29) – subcutaneous progesterone 25 mg once daily, group 3 (n = 25) – micronized vaginal progesterone 200 mg 3 times daily. LPS was prescribed daily starting from the day of transvaginal ovarian puncture. At pregnancy onset, gestagens were continued until 12 weeks’ gestation. The primary efficacy endpoints were the rates of biochemical and clinical pregnancies, and the secondary endpoints were the rates of missed miscarriage and vaginal bleeding during early pregnancy. Results. The rates of biochemical pregnancy, clinical pregnancy, and missed miscarriage were comparable in the groups of oral dydrogesterone (38.00%), subcutaneous progesterone (37.93%) and micronized vaginal progesterone (36.00%), no statistically significant difference was found. There was a trend towards a lower risk of vaginal bleeding in the dydrogesterone (12.00%) and subcutaneous progesterone (13.79%) groups compared to the micronized vaginal progesterone group (24.00%), but the difference was not statistically significant (p = 0.694). Conclusion. Any of the presented forms of gestagens can be used in clinical practice, as the efficacy in all groups was comparable, with no statistically significant difference between them. Key words: dydrogesterone, progesterone, assisted reproductive technologies, in vitro fertilization, luteal phase support

Sperm agglutination and ejaculate liquefaction time as negative prognostic factor in ICSI

Background. Not all pathological mechanisms of fertility disorders in men are currently the correct diagnostic tests, this significantly reduces the prognostic value of the spermogram and makes the question of assessing spermogram character disorders and its impact on the effectiveness of assisted reproductive technologies (ART) procedures extremely relevant. In our study, we retrospectively evaluated the parameters of ejaculate and their effect on ART outcomes.Aim. To assess the effect of sperm agglutination and ejaculate liquefaction time on in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) results.Materials and methods. The selection of married couples was carried out on the basis of the V.I. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology of the Ministry of Health of Russia. The study included 6,416 married couples who sought IVF-ICSI in the period from December 2012 to December 2016. Ages of men varied between 23 and 74 years, ages of women between 17 and 53 years. Examination data of the patients were extracted from the BABE software. In the study, sperm analysis parameters were evaluated during preparation to the IVF program, IVF-ICSI, and on the day of transvaginal ovarian puncture. Using univariant and regression analysis, prognostic factors of clinical pregnancy and childbirth after IVF-ICSI were determined.Results. Sperm agglutination rates adversely affect the incidence of clinical pregnancy (probability ratio (PR) 0.701; 95 % confidence interval (CI) 0.589–0.834; p &lt;0.001) and childbirth (PR 1.719; 95 % CI 1.220–2.422; p = 0.002) in IVF-ICSI assisted reproductive technologies. Ejaculate liquefaction has a negative effect on the incidence of clinical IVF-ICSI pregnancy (PR 0.967; 95 % CI 0.949–0.986; p = 0.001).Conclusion. Andrologists should seek to improve the above ejaculate parameters with various treatments to improve the effectiveness of ART programs.

Higher Doses of FSH Used for Superovulation Do Not Adversely Affect Embryonic Ploidy: A Randomised Controlled Trial (Stimulation Resulting in Embryonic Aneuploidy Using Menopur (STREAM) Trial)

Background: High dose gonadotropin stimulation protocols may increase costs, patient discomfort, likelihood of ovarian hyper stimulation syndrome (OHSS), ovarian torsion and increased bleeding after multiple ovarian punctures. In addition, it is possible that high dose stimulation may result in impaired oocyte “quality” with adverse effect of embryonic ploidy. This may negate any benefits seen from a higher oocyte yield after high dose stimulation. Aim: To determine the proportion of euploid blastocysts resulting from higher versus lower dose ovarian stimulation protocols. Method: Multicentre randomised controlled trial recruiting 57 women who were treated with ovarian stimulation using either 150 or 300 IU Menopur per day. In Vitro Fertilisation (IVF) or Intracytoplasmic Sperm Injection (ICSI) were performed according to individual unit protocol and embryos were cultured to blastocyst stage. A trophectoderm biopsy was performed for preimplant action genetic testing for aneuploidy. Results: The number of oocytes obtained from women randomised to 150 IU Menopur was between 3–17 (mean=9), whereas the number of oocytes obtained from women randomised to 300 IU Menopur was between 3–24 (mean=11). There was a positive linear relationship between serum AMH concentration and oocyte yield in both the 150 &amp; 300 IU Menopur groups (R=0.3359, R2=0.1129 &amp; R=0.3741, R2=0.1399). 63% of blastocysts were euploid in the 150 IU Menopur group and 75% in the 300 IU Menopur group (p=0.17). Conclusion: The higher dose ovarian stimulation protocol did not significantly increase the number of oocytes retrieved, nor did the higher dose protocol reduce the proportion of euploid embryos created. This study does not support the hypothesis that use of higher doses of gonadotropin for ovarian stimulation results in a reduction in the proportion of euploid embryos obtained after IVF.

TGFß Gene Members and Their Regulatory Factors in Granulosa Compared to Cumulus Cells in PCOS: A Case-Control Study.

Objective Transforming growth factor-beta (TGF-β) superfamily and its members that include bone morphogenetic protein 15 (BMP15), anti-Mullerian hormone (AMH), growth /differentiation factor-9 (GDF9), and their respective receptors: BMPR1A, BMPR1B, and BMPR2 have been implicated as key regulators in various aspects of ovarian function. The abnormal function of the ovaries is one of the main contributing factors to polycystic ovarian syndrome (PCOS), so this study aimed to investigate the mRNA expression profile of these factors in granulosa (GCs) and cumulus cells (CCs) of those patients. Materials and Methods The case-control research was conducted on 30 women (15 infertile PCOS and 15 normo-ovulatory patients, 22≤age ≤38 years old) who underwent ovarian stimulation for in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycle. GCs/CCs were obtained during ovarian puncture. The expression analysis of the aforementioned genes was quantified using real-time polymerase chain reaction (PCR). Results AMH and BMPR1A expression levels were significantly increased in GCs of PCOS compared to the control group. In contrast, GDF9, BMP15, BMPR1B, and BMPR2 expressions were decreased. PCOS' CC showed the same expression patterns. GDF9 and AMH were effectively expressed in normal CCs, and BMP15 and BMPR1B in normal GCs (P<0.05). ConclusionDifferential gene expression levels of AMH and its regulatory factors and their primary receptors were detected in granulosa and cumulus cells in PCOS women. Since the same antagonist protocol for ovarian stimulation was used in both PCOS and control groups, the results were independent of the protocols. This diversity in gene expression pattern may contribute to downstream pathways alteration of these genes, which are involved in oocyte competence and maturation.

P-591 Cellular heterogeneity in the preovulatory follicle correlates with response to stimulation with recombinant FSH and affects somatic cell gene expression levels

Abstract Study question What is the difference in the composition and gene expression of pre-ovulatory follicular somatic cells between normo- and hyporesponder patients to rFSH stimulation? Summary answer Gene expression changes in the somatic cells of the preovulatory follicles of hyporesponder patients is partly attributable to distinct cell populations. What is known already Some women undergoing IVF respond suboptimally to the preceding ovarian stimulation without an indication of poor ovarian insufficiency nor advanced age. The gene expression levels in the somatic cells of their pre-ovulatory follicles have not been determined. However, the somatic cells play key roles in the transfer of gonadotrophin signals and in steroidogenesis. In the preovulatory follicle, being &amp;gt;20 mm in size, the somatic cells are exposed to various molecular gradients that drive their differentiation. Due to advancements in sequencing technologies the importance of somatic cell sub-populations in the etiologies of ovarian infertility can now be investigated by bioinformatic methods. Study design, size, duration Consecutive IVF patients &amp;lt;41 years of age were recruited at Nova Vita Clinic, Estonia, undergoing the antagonist stimulation protocol with rFSH administration. Ovarian puncture was performed for all patients after 36 hours of hCG stimulaton. rFSH dose &amp;gt;200 IU per one retrieved oocyte was considered as an indication for hyporesponse. Ten normo- and 9 hyporesponder patients were enrolled. Participants/materials, setting, methods Cells from the follicular fluid devoid of the cumulus oocyte complex were collected by centrifugation. RNA-seq was performed on the pooled cells of all participants. The study groups were compared by differential gene expression analysis by DESeq2. Single-cell RNA-seq was performed for &amp;gt;6000 individual cells of 3 normoresponder patients and cellular sub-populations were determined with Seurat package. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data of all participants. Main results and the role of chance Alterations in the gene expression of ovarian somatic cells have been previously attributed to female age. We demonstrate that 407 genes are differentially expressed (FDR&amp;lt;0.05) between normo- and hyporesponder IVF patients after age adjustment. These genes were enriched into pathways of extracellular matrix reorganization (31 genes, FDR&amp;lt;0.005), post-translational protein phosphorylation (16 genes, FDR&amp;lt;0.006) and regulation of insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins (16 genes, FDR&amp;lt;0.019). In addition, the sequencing of &amp;gt; 24 000 individual cells from 3 normoresponder patients revealed the somatic cell types of the preovulatory follicle in high resolution indicating the presence of immune cell populations (N = 4), epithelial cells, theca, cumulus cells and mural granulosa cell sub-populations (N = 7). Bioinformatic cell type deconvolution demonstrated significant differences between proportions of distinct cell sub-populations between normo- and hyporesponder patients and that several differentially expressed genes between the study groups can be attributed to a specific mural granulosa sub-population. Limitations, reasons for caution The number of study participants is small due to the high cost of the study methods. Wider implications of the findings The study demonstrates that hyporesponse to stimulation is associated with age-unrelated disturbances in gene expression of preovulatory somatic cells and that different somatic cell populations may have important underlying functions in the ovarian etiologies of infertility. Trial registration number not applicable

P-634 Safety of ART cycles with extremely high estradiol levels - A retrospective cohort study

Abstract Study question Are IVF treatments with extremely high peak estradiol levels during ovarian hyperstimulation associated with higher complication rates? Summary answer Extremely high estradiol levels were not associated with higher major complication rates, but patients were more likely to seek medical care following retrieval. What is known already The freeze-all policy and the increasing popularity of social oocyte freezing, have led to an increase in gonadotropin doses and number of oocytes retrieved and, as a direct effect, higher estradiol levels. Studies reporting major ART complications (OHSS, bleeding, infection, torsion, thromboembolic events) are based on heterogeneous patient populations without stratification by oocyte number or estradiol levels, except for a single study reporting an association between peak estradiol levels and the risk of ovarian torsion. Extreme estradiol levels, are associated with larger ovaries, repeated ovarian punctures, and possibly a thrombogenic effect. Therefore, higher complication rates can be expected. Study design, size, duration A retrospective cohort study including patients from two large medical centers treated between 2019-2021. Participants/materials, setting, methods A study group (239 patients) with extremely high peak estradiol levels (&amp;gt;20,000 pmol/L on the day of ovulation induction, or &amp;gt; 15,000 pmol/L on the previous day) and a control group (208 patients) with normal range estradiol levels (3000-12000pmol/L), treated at two large units were included. Patients were surveyed about complaints and medical care related to ovum pick up (OPU), and medical files were reviewed. Complication rates and the need for medical assistance were compared. Main results and the role of chance Several differences between the study and control group were observed as a consequence of the study design: Mean age was 33.01±5.14 vs. 34.57±4.52 (p = 0.01), Mean peak estradiol levels was 26645.34±8592.56 vs.7229.750±2329.20 (p &amp;lt; 0.001), mean number of oocytes were 27.55±13.46 vs. 11.63±5.77 (p &amp;lt; 0.001) for the study and control group respectivly. More patients in the control group underwent fertility preservation. Mean FSH levels and total gonadotropin dosage were higher in the control group. Major complications [3 (1.25%) in the study group vs. 1 (0.96%) in the control group] were similar between groups (p = 0.62). In the study group Two patients were diagnosed with ovarian torsion (0.83%), and one (0.41%) with early severe OHSS as a result of a positive BhCG related to an undiagnosed pregnancy prior to OPU. All three were admitted (1.25%). In the control group one patient was diagnosed with PID requiring inpatient IV antibiotic treatment (0.48%). One more patient was admitted with severe abdominal pain following OPU, for a total of 2 admissions (0.96%) (p = 1). 33 patients (13.8%) in the study group and 10 (4.8%) in the control group sought medical care after OPU, mostly due to abdominal pain, without further workup or hospitalization (p = 0.001). Limitations, reasons for caution A retrospective study with possible recall bias. Major adverse events are rare in IVF and may not be fully captured in the study population. Wider implications of the findings Based on our results, extremely high estradiol levels during ovarian hyperstimulation were not associated with thromboembolic events, higher major complication or hospitalization rates, thus may be considered safe. Nevertheless, patients may be informed of possible higher rates of discomfort, mostly abdominal pain. Larger studies are warranted to confirm our results. Trial registration number 0090-21-ASF

Effect of Transvaginal Aspiration of Oocytes on Blood And Peritoneal Fluid Parameters in Mares

Transvaginal aspiration of oocytes (TVA) is a commonly used clinical procedure to obtain oocytes for in vitro production of embryos in horses. The goal of this study was to evaluate the effect of the TVA procedure on blood and peritoneal parameters, and to investigate the association of these findings with variables such as use of antibiotics, number of ovarian punctures, and length of the procedure. Physical examination was performed and blood and peritoneal fluid were obtained from 14 mares before they underwent TVA and the same parameters were assessed 24 hours after the procedure. On examination, 13/14 mares remained clinically healthy after the procedure. One mare developed low-grade fever, transient anorexia and mild colic that resolved within 12 hours post-TVA. The use of antibiotics, length of procedure and number of ovarian punctures did not have an effect on the measured parameters. All the mares presented significant changes in the leukogram, but these mostly remained within normal reference range values. The peritoneal parameters were also consistently affected after TVA. A significant post-TVA increase in lactate, total protein, and peritoneal neutrophil count was observed in peritoneal fluid. Peritoneal lactate level was elevated above baseline physiological levels in more than 50% of the mares. Results from this study indicate that there is an expected degree of inflammation after TVA procedures and peritoneal fluid parameters could be successfully used to monitor inflammation in the early stages.

Progesterone-induced blocking factor 1 and cytokine profile of follicular fluid of infertile women qualified to in vitro fertilization: The influence on fetus development and pregnancy outcome.

Introduction: Progesterone is essential for both the initiation and the maintenance of pregnancy. The immunological effects of progesterone are mediated by the progesterone-induced blocking factor (PIBF), which is an immunomodulatory factor with anti-abortive properties. The aim of the research was to establish the cytokine profile and PIBF1 concentration in follicular fluid (FF) of patients undergoing in vitro fertilization (IVF).Methods: Seventy-eight patients who qualified for IVF underwent a detailed medical interview, including the course of fertility treatment and physical, gynecological, and cytological examinations. The concentration of PIBF1, IL-18, IL-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), IL-1α, IL-1β, IL-5, IL-8, and IL-15 in FF during ovarian puncture was measured using commercially available ELISA kits.Results: IL-1 beta concentration was lower in the FF of patients with successful IVF. IL-8 concentration in FF correlated with the number of cumulus-oocyte complexes (COC-1), metaphase II (MII), and top-quality embryos. PIBF1 concentration had a positive correlation with the number of MII and top-quality embryos. IL-2 and IL-6 concentrations were positively correlated with the number of COC-1 and MII. An important parameter in assessing the chances of successful IVF is the number of top-quality embryos achieved.Conclusion: Higher PIBF1 concentration in FF may indicate a greater possibility of successful IVF due to the higher number of top-quality embryos. IL-1 beta concentration was found to be lower in the FF of patients with successful IVF. Therefore, PIBF1 and IL-1 beta in FF could be candidates for a marker of successful IVF.

The Estimation Of The Quantity And Quality Of Oocytes And Embryos In The IVF Protocols In Women With Uterine Fibroids With And Without Surgical Treatment

Myoma periodically leads to infertility by causing impaired blood flow, aseptic inflammation in the endometrium, increased uterine contractility, impaired sperm migration with cerviсal myoma nodes and obstruction of the proximal fallopian tubes. Objective — The estimation of the quantity and quality of oocytes and embryos in the IVF (in vitro fertilization) protocols in women with uterine fibroids with and without surgical treatment. Material and Methods — The retrospective study included 37 IVF cycles from 37 patients that had infertility combined with myoma. The Group 1 patients were subjected to the IVF procedure without previous treatment of the myoma, while the Group 2 patients were subjected to IVF after myomectomy. Results — The total quantity of oocytes and the percentage of mature (MII) oocytes obtained during the ovarian puncture of the patients in the compared groups were slightly higher in the group without surgical treatment (p&gt;0.05). Any significant differences in the frequency of formation of high-quality embryos (р=0.43) and good-quality embryos (р=0.22) between the groups were not obtained. The number of embryos suitable for embryo transfer to the uterine cavity and/or cryopreservation in the women with myomectomy was 9% higher than the one in the group without surgical treatment. Conclusion — Surgical treatment of uterine fibroids does not significantly affect the quantity and quality of oocytes and embryos obtained in the IVF procedure.

Effect of intrauterine granulocyte-colony stimulating factor administration on in vitro fertilization outcome in women with moderate-to-severe endometriosis: An RCT.

BackgroundNearly 25-50% of infertile women have endometriosis. There are reports of disorders in the expression of granulocyte colony-stimulating factor (G-CSF) receptors in women with endometriosis.ObjectiveTo examine the effect of intrauterine administration of G-CSF in in vitro fertilization (IVF) cycles on the fertility rate of infertile women with moderate-to-severe endometriosis.Materials and Methods This clinical trial was conducted on 66 infertile women with moderate-to-severe endometriosis, undergoing IVF and intracytoplasmic sperm injection (ICSI). The participants were allocated into two groups via simple randomization: the G-CSF (n = 27) and control (n = 39) groups. In the G-CSF intervention group, on the oocyte pick-up day, immediately after an ovarian puncture, 300 μg of G-CSF was injected through a transcervical catheter under abdominal ultrasound guide to visualize flushing into the uterine cavity. Women in the control group received no intervention. The two groups were evaluated for clinical pregnancy.ResultsNo significant difference was noted in the demographic characteristics of the two groups. The rate of clinical pregnancy was 28.2% in the control group and 25.9% in the G-CSF group, indicating no significant difference (p = 0.83).ConclusionThe results showed that the intrauterine injection of G-CSF had no effects on pregnancy in women with stage-3/4 endometriosis undergoing IVF.

P–689 IL–6/IL–10 ratio as predictor of poor ovarian response in women undergoing in-vitro fertilization

Abstract Study question Are serum cytokines levels associated with ovarian response in IVF cycles? Summary answer The IL–6/IL–10 ratio is higher in patients with low ovarian response. What is known already Previous studies reported differences in the levels of IL–2, Il–6, IL–8, IL–10 and VEGF in follicular fluid between young patients with low ovarian response and normoresponder women. In addition, it is known that IL–6 plays an important role as a mediator of fever and acute phase reaction and IL–10 is the cytokine with the greatest anti-inflammatory power. Although there seems to be some evidence about the possible effect of the immune system on ovarian function and implantation, the role it plays in ART remains unknown. Our aim was to investigate the effect of cytokines in ovarian reserve and response. Study design, size, duration One hundred and fifty-two patients were included in a retrospective study between February 2016 and December 2020. Serum cytokines IL–2, IL–4, IL- 6, IL–8, IL–10, VEGF, IFN↖, TNF α, IL–1 α, IL–1 β, MCP–1 and EGF were measured previously to the ovarian stimulation cycle. Patients with altered karyotype, mutation or premutation in the FMR1 gene or endometriosis or with any other factor that could alter the ovarian reserve or response were excluded from the study. Participants/materials, setting, methods To measure the levels of the different cytokines, a sandwich immunoassay with specific antibodies for the cytokines IL–2, IL–4, IL–6, IL–8, IL–10, VEGF, IFN↖, TNF α, IL -1 α, IL–1 β, MCP–1 and EGF were used. The statistical analysis was performed with R Statistical Software, version 4.0.3 and the Software Statistical Product and Service Solutions, version 20.0 (SPSS, Chicago, IL, EE.UU.). Main results and the role of chance We found that the ratio between IL–6 and IL–10 cytokines is higher in those patients in whom four or fewer oocytes have been recovered after ovarian puncture (2.15 versus 1.55; p = 0.035; Mann-Whitney test). If we establish 0.9 as a cut-off point for the IL–6 / IL–10 ratio, we observed that above this value the risk of having a low response to ovarian stimulation is more than 3 times greater than below this value (22.9% versus 6.0%; p = 0.007; Fischer exact test). There were no statistically significant differences between both groups in terms of age (p = 0.136), dose of gonadotropin administered (p = 0.415) and duration of ovarian stimulation (p = 0.706). In addition, performing hierarchical cluster analysis with the analyzed cytokines and the associated variables to ovarian reserve and response, we observed that the antral follicle count, the total oocytes recovered and the MII recovered are grouped in the same cluster as the cytokines IL–2, IL–4, IL–6, IL–10, IL–1α, IL–1B, IFNγ y TNFα. We determined the number of clusters based on the tree diagram and k-means method. Limitations, reasons for caution The retrospective study design and the sample size could be a limitation. The study was performed in patients with suspected implantation failure. Wider implications of the findings: The ratio between IL–6 and IL–10 could be used as a potential biomarker to predict the ovarian response and provide real expectations regarding the success of IVF cycle. The action of IL–6 could be reduced by blocking its receptor using humanized monoclonal antibodies as Tocilizumab. Trial registration number Not applicable