Showing papers by "Susanne Elisabeth Pors published in 2022"

Intrafollicular Concentrations of the Oocyte-secreted Factors GDF9 and BMP15 Vary Inversely in Polycystic Ovaries

Abstract: Abstract Context The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome. Objective Are there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)? Design and Setting Follicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation. Patients Twenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size. Main outcome measures Intrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays. Results The detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ± 189 pg/mL [mean ± SEM], n = 188; non-PCO: 3498 ± 199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ± 40 pg/mL, n = 125; non-PCO: 573 ± 55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO. Conclusions Intrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO.

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue

Abstract: Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

Oocyte diameter predicts the maturation rate of human immature oocytes collected ex vivo

Abstract: Abstract Purpose To study the impact of oocyte diameter and cumulus cell mass on the potential for final maturation of immature human oocytes in vitro. Methods Immature oocytes ( n = 1563) from 75 women undergoing fertility preservation by ovarian tissue cryopreservation (14–41 years) were collected. After preparation of the ovarian cortex for freezing, immature oocytes were collected from the surplus medulla. After collection, IVM was performed according to standard published methods. The mass of cumulus cell surrounding the immature oocyte was grouped according to size. After IVM, each oocyte was photographed, measured, and the diameter was calculated as a mean of two perpendicular measurements. Results The diameter of the oocytes ranged from 60 to 171 µm with a mean of 115 µm (SD:12.1) and an interquartile range from 107 to 124 µm. The oocyte diameter was positively associated with a higher incidence of MII ( p < 0.001). MII oocytes had a significantly larger mean diameter than MI, GV, and degenerated oocytes. The size of the cumulus cell mass was significantly associated with the MII stage ( p < 0.001) and larger oocyte diameter ( p < 0.001). The results further confirm that the diameter of the fully grown oocyte is reached relatively early in human follicular development and that the factors governing oocyte maturation in vitro are connected to the surrounding cell mass and the oocyte. Conclusion The diameter of the oocyte is a highly determining factor in the nuclear maturation of the human oocyte during in vitro maturation, and the size of the cumulus cell mass is closely positively associated with a larger diameter.

Revascularization of human ovarian grafts is equally efficient from both sides of the cortex tissue.

Abstract: Does revascularization of human ovarian grafts in a mouse model occur with equal efficiency from both sides of the cortex tissue?Twenty-four frozen-thawed ovarian cortex pieces from 12 women were transplanted to immunodeficient mice, for 8 days to analyse graft revascularization using immunohistochemical detection of murine CD31, or for 8 weeks to evaluate follicle density (follicles/mm3). The CD31-positive vessel area and density were quantified using a custom-designed application. Three regions of interest (ROI) were defined in each tissue section: the cortical side, the centre and the medullary side. Vessels were subdivided into three categories according to size: microvessels (<300 µm2), small vessels (300-1000 µm2) and large vessels (>1000-3000 µm2).No significant difference in the mean percentage of the CD31-positive vessel area was found between the three ROI (cortical side: 3.9% ± 0.2%; centre: 3.5% ± 0.2%; medullary side: 4.0% ± 0.3%; P = 0.17), but a significantly lower density of vessels was found in the centre of the human ovarian grafts compared with the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; centre: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; P < 0.001). Microvessels comprised 89-91% of all vessels in the three ROI. Follicle density in ungrafted cortex pieces was 51.8 ± 17.3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks of xenografting, resulting in a follicle survival rate of 28%.Host revascularization was established equally efficiently from both sides of transplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft could potentially facilitate faster graft revascularization.

Organotypic Culture of Testicular Tissue from Infant Boys with Cryptorchidism

Abstract: Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression (p < 0.001) and decreased AMH expression (p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells (p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced (p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium (p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.

Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation

Abstract: Background Cryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications. Methods Testis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1). Results Following xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes. Conclusion Xenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.

Concentrations of oocyte secreted GDF9 and BMP15 decrease with MII transition during human IVM

Abstract: Abstract Background The suggested effects of the oocyte secreted GDF9 and BMP15 growth factors on oocyte maturation are currently based on recombinant proteins, and little is known about native GDF9 and BMP15 in humans. Methods Human immature cumulus-oocyte complexes (COCs) obtained in connection with ovarian tissue cryopreservation (OTC) underwent in vitro maturation (IVM). Oocyte-produced GDF9 and BMP15 were detected in COCs using immunofluorescence, and in fresh GV oocytes and in GV and MII oocytes after IVM by western blot. Concentrations of GDF9, BMP15 homodimers, and GDF9/BMP15 heterodimer in spent media after IVM were measured by ELISA. The relative expression of seven genes from the GDF9 and BMP15 signaling pathways ( BMPR2, ALK5, ALK6, SMAD1, SMAD2, SMAD3, and SMAD5 ) was evaluated in fresh cumulus cells (before IVM) and in cumulus cells from GV and MII oocytes after IVM by RT-qPCR. Results We detected native pro-mature GDF9 and BMP15 in human oocytes with molecular weights (Mw) of 47 kDa and 43 kDa, respectively. Concentrations of GDF9 and BMP15 in spent media after IVM were detected in 99% and 64% of the samples, respectively. The GDF9/BMP15 heterodimer was detected in 76% of the samples. Overall, the concentration of GDF9 was approximately 10-times higher than BMP15. The concentrations of both GDF9 and BMP15 were significantly lower in spent medium from MII oocytes than in media from oocytes that remained at the GV stage. Concentrations of the GDF9/BMP15 heterodimer did not differ between GV and MII oocytes. Furthermore, BMPR2 , SMAD3 , and SMAD5 were significantly upregulated in cumulus cells from MII oocytes, indicating that both GDF9 and BMP15 signaling were active during oocyte meiotic resumption in vitro . Conclusion These data suggest that the driving mechanisms for oocyte nuclear maturation may involve both GDF9 and BMP15 homodimers, while the role of the GDF9/BMP15 heterodimer is questionable.

Sertoli and Germ Cells Within Atrophic Seminiferous Tubules of Men With Non-Obstructive Azoospermia

Abstract: Background Infertile men with non-obstructive azoospermia (NOA) have impaired spermatogenesis. Dilated and un-dilated atrophic seminiferous tubules are often present in the testes of these patients, with the highest likelihood of active spermatogenesis in the dilated tubules. Little is known about the un-dilated tubules, which in NOA patients constitute the majority. To advance therapeutic strategies for men with NOA who fail surgical sperm retrieval we aimed to characterize the spermatogonial stem cell microenvironment in atrophic un-dilated tubules. Methods Testis biopsies approximately 3x3x3 mm3 were obtained from un-dilated areas from 34 patients. They were classified as hypospermatogenesis (HS) (n=5), maturation arrest (MA) (n=14), and Sertoli cell only (SCO) (n= 15). Testis samples from five fertile men were included as controls. Biopsies were used for histological analysis, RT-PCR analysis and immunofluorescence of germ and Sertoli cell markers. Results Anti-Müllerian hormone mRNA and protein expression was increased in un-dilated tubules in all three NOA subtypes, compared to the control, showing an immature state of Sertoli cells (p<0.05). The GDNF mRNA expression was significantly increased in MA (P=0.0003). The BMP4 mRNA expression showed a significant increase in HS, MA, and SCO (P=0.02, P=0.0005, P=0.02, respectively). The thickness of the tubule wall was increased 2.2-fold in the SCO-NOA compared to the control (p<0.05). In germ cells, we found the DEAD-box helicase 4 (DDX4) and melanoma-associated antigen A4 (MAGE-A4) mRNA and protein expression reduced in NOA (MAGE-A: 46% decrease in HS, 53% decrease in MA, absent in SCO). In HS-NOA, the number of androgen receptor positive Sertoli cells was reduced 30% with a similar pattern in mRNA expression. The γH2AX expression was increased in SCO as compared to HS and MA. However, none of these differences reached statistical significance probably due to low number of samples. Conclusions Sertoli cells were shown to be immature in un-dilated tubules of three NOA subtypes. The increased DNA damage in Sertoli cells and thicker tubule wall in SCO suggested a different mechanism for the absence of spermatogenesis from SCO to HS and MA. These results expand insight into the differences in un-dilated tubules from the different types of NOA patients.

O-037 Revascularization of human ovarian grafts occurs equally efficient from both sides of the cortex tissue: implications for clinical practice

Abstract: Does revascularization of xenotransplanted human ovarian grafts occur equally efficient from both the cortical and medullary side of the cortex tissue? Murine vessels were established equally efficient from both sides of human ovarian grafts, demonstrating that the fibrous cortical side does not hinder the neovascularization process. There is a lack of consensus regarding the surgical approach for transplanting frozen-thawed ovarian tissue and various techniques are being used clinically worldwide. Some centers attach ovarian tissue with stitches or Interceed® on top of the decorticated remaining ovary to mimic normal ovarian architecture. Other centers transplant ovarian tissue in subcortical pockets made by longitudinal or transverse incisions in the remaining intact ovary to facilitate vascularization from both sides of the graft. While other centers transplant to sub-peritoneal pelvic sites only. It is unknown which techniques and transplantation sites provide the most efficient revascularization of the ovarian grafts. Three pieces of cryopreserved ovarian cortex (approximately 5x5x1 mm in size) were donated and thawed from 12 women and two of the pieces were transplanted to subcutaneous pockets on the dorsal side of ovariectomized immunodeficient female mice. The third piece was processed as an ungrafted control. Ovarian grafts were retrieved after 8 days to analyze the spatial distribution of graft-revascularization using immunohistochemical detection of murine CD31, and after 8 weeks to evaluate follicle density (follicles/mm3). Ovarian cortex was donated from 12 women aged 27-31 years having their tissue frozen for fertility preservation. The CD31 positive vessel area and density were quantified using a custom designed application (APP) from Visiopharm®. Three regions of interest (ROIs) were defined in each tissue section; the cortical side, the center, and the medullary side. Vessels were sub-divided into three categories according to size: micro-vessels (<300 µm2), small vessels (300-1000 µm2), and large vessels (1000-3000 µm2). After 8 days xenotransplantation, a statistical significant lower density of vessels was found in the center of the human ovarian grafts compared to the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; center: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; p < 0.001). The mean percentage of CD31 positive vessel area in the human ovarian grafts was similar in the three ROIs, but lowest in the center of the grafts (cortical side: 3.9% ± 0.2 (SEM); center: 3.5% ± 0.2; medullary side: 4.0% ± 0.3; p = 0.17). Micro-vessels comprised 89-91% of all vessels in the three ROIs, demonstrating that the vast majority of vessels were newly formed. Heatmaps were generated for each section based on the CD31 positive area to visualize the localization of vessels in the ovarian grafts and the maps showed highly variable vascularization patterns in the grafts from different patients. Overall, vascularization appeared to occur equally efficient in all peripherical areas of the ovarian grafts. The follicle density in ungrafted cortex tissue was 51.8 ± 17.3 follicles/mm3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks xenografting, resulting in an average follicle survival rate of 28%. The current study was not designed to directly compare clinically used transplantation techniques, which should be studied in larger animal models, like sheep, in which the structure of ovarian tissue is similar to human. Revascularization was established equally efficient from both sides of xenotransplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft, such as sub-cortical or peritoneal pockets, may facilitate a more efficient revascularization of the graft than techniques allowing revascularization from only the medullary side. not applicable

Proteomic Alterations in Follicular Fluid of Human Small Antral Follicles Collected from Polycystic Ovaries—A Pilot Study

Abstract: Polycystic ovaries (PCO) contain antral follicles that arrest growing around 3–11 mm in diameter, perturbing the dominant follicle’s selection and the subsequent ovulatory process. Proteomic alterations of PCO follicular fluid (FF) (i.e., microenvironment in which the oocyte develops until ovulation) have been studied from large follicles in connection with oocyte pickup during ovarian stimulation. The present study aimed to detect proteomic alterations in FF from unstimulated human small antral follicles (hSAF) obtained from PCO. After performing deep-sequencing label-free proteomics on 10 PCO and 10 non-PCO FF samples from unstimulated hSAF (4.6–9.8 mm), 1436 proteins were identified, of which 115 were dysregulated in PCO FF samples. Pathways and processes related to the immune system, inflammation, and oxidative stress appeared to be upregulated in PCO, while extracellular matrix receptors interactions, the collagens-containing extracellular matrix, and the regulation of signaling were downregulated. The secreted proteins SFRP1, THBS4, and C1QC significantly decreased their expression in PCO FF, and this downregulation was suggested to affect future oocyte competence. In conclusion, our study revealed, for the first time, evidence of proteomic alterations occurring in the FF of PCO hSAF that may be related to the dysfunction of follicular growth and subsequent oocyte competence.

P-450 Mechanical injury improves follicle survival in xenotransplanted human ovarian cortex

Abstract: Can mechanical injury by needle puncturing of frozen thawed human ovarian cortex tissue prior to transplantation increase revascularization of xenografts and improve follicle survival? Mechanical injury caused by needle puncture prior to transplantation improved follicle survival 2-fold in human ovarian cortex tissue after 4 weeks xenografting. Cryopreservation and autotransplantation of ovarian tissue is an established fertility preservation method which has resulted in numerous live births worldwide. However, the efficiency of the method is hampered by a critical follicle loss after transplantation. Faster revascularization of the transplanted graft would shorten the ischemic period and could potentially improve follicle survival. Controlled tissue damage by laser or needles can induce local angiogenesis by initiating the wound healing mechanisms and has been applied clinically in cardiovascular surgery for decades. Whether controlled mechanical tissue damage could be used to improve grafting of human ovarian cortex tissue has not yet been investigated. A total of 32 frozen-thawed human ovarian cortex pieces were cut in halves; one half serving as the untreated control and the other half was punctured approximately 200 times with a 29G needle immediately after thawing. The cortical pieces were transplanted subcutaneously to 10 immunodeficient mice. Ovarian grafts were retrieved after 3, 6 and 10 days (n = 8) to analyse graft revascularization and gene expression, and after 4 weeks to assess follicle density (n = 8). Ovarian cortex was donated by 16 women (aged 24-36 years) undergoing ovarian tissue cryopreservation for fertility preservation at a University Hospital. Gene expression analysis of proinflammatory cytokines (Tnf-α), apoptotic factors (Bcl2 and Bax), and angiogenic factors (Vegfa, Angptl4, Ang1, and Ang2) were performed by qPCR. Follicle density after 4 weeks xenografting were evaluated histologically. Graft-revascularization was assessed using immunohistochemical detection of murine Cd31. Our results showed that the ovarian grafts which had been punctured with needles prior to transplantation contained a larger number of surviving follicles per mm3 compared to the untreated control (control group: 2.9 ± 0.6 (mean±SEM) follicles/mm3; needle group: 6.4 ± 2.4 follicles/mm3). Follicle densities between patients and groups varied from 0.8 to 26 follicles/mm3. Overall, a 2.3-fold increase in follicle density was found in the needle group compared to control, though this finding was not significantly different (p-value = 0.1182). The relative gene expression of Tnf-α and the Bax/Bcl2 ratio after 6 days grafting was similar in the needle and control group, indicating that the mechanical injury did not induce an additional inflammatory or apoptotic response in the grafts. The relative gene expression of Vegf, Angptl4, and Ang2 after 6 days grafting was highest in the needle group compared to the control group, though not significant (p>0.1). Results of the gene expression analysis from Day 3 and 10 are awaited, and so are the quantification of the Cd31 positive vessel area and vascular density from Day 3, 6, and 10, which will provide valuable insight into the initial graft revascularization. The ovarian follicles are highly heterogeneously distributed in the ovarian cortex and can vary 100-fold between cortex pieces from the same woman and between patients. A high variability in follicle density within and between treatment groups and patients was found in the current study. Thus, solid conclusions cannot be made. Our findings suggest a positive effect of mechanical injury on human ovarian grafts as needle puncturing prior to transplantation improved follicle survival after xenotransplantation. Clinically, this technique would be easy to implement since it is performed during the thawing process and does not involve any additional treatment of the patient. Not applicable

P-626 Eight weeks of androgen priming by low dose hCG before IVF/ICSI in women with low ovarian reserve

Abstract: Does eight weeks of low dose hCG priming improve the outcome of ovarian stimulation for IVF/ICSI in women with low ovarian reserve. Significantly more oocytes were retrieved following eight weeks of low dose hCG priming, when compared to an identical IVF/ICSI treatment immediately before the priming period. Administration of androgens prior to ovarian stimulation for IVF might upregulate the FSH receptor level on granulosa cells making the follicles more responsive to exogenous FSH. LH and hCG stimulate the follicular androgen synthesis, acting through LH receptors on the theca cells, enabling their use as an intra-ovarian androgen priming method. Exogenous testosterone and DHEA priming have been shown to increase the number of retrieved oocytes and the live birth rate, but systemic androgen administration is associated with side effects. Long term ‘intra-ovarian’ priming with hCG without systemic effects in women with low ovarian reserve has not previously been investigated. A prospective, paired, non-blinded study including 20 women serving as their own controls conducted between January 2021 and July 2021 at The University Hospital Copenhagen Rigshospitalet, Denmark. Participants underwent two identical consecutive IVF/ICSI treatments, a Control cycle including elective freezing of all blastocysts and a Study cycle with fresh blastocyst transfer. The Control and Study cycle were separated by approximately eight weeks (two menstrual cycles) of androgen priming by daily injections of 260 IE hCG. Inclusion criteria were age 18-40 years, regular menstrual cycle 23-35 days and AMH <6.29 pmol/L. Control and Study IVF/ICSI cycles were performed in a fixed GnRH-antagonist protocol using a daily dose of 300 IU rFSH and GnRH antagonist 0.25 mg from stimulation day 5-6. Primary endpoint was the follicular output rate defined as the pre-ovulatory follicle count (>16 mm) on hCG trigger day divided by the antral follicle count (2-10 mm) on cycle day 2-3. 20 women with a mean (SD) age of 36.8 (3.2) years and an AMH level of 3.7 (1.5) pmol/L were included and completed the study. The number of oocytes retrieved was significantly higher in the Study compared to Control Cycle: 4.7 (2.8) vs. 3.2 (1.7), (P = 0.007), and the number of blastocysts was 1.2 (1.6) vs. 0.6 (0.7) in the Study compared to Control cycle, (P = 0.11). The follicular output rate was 0.4 (0.4) in the Study cycle initiated immediately after eight weeks of hCG priming, which was similar to the follicular output rate of 0.38 (0.2) in the Control cycle performed just before initiating the priming period (P = 0.81). The number of follicles above 10 mm on trigger day was 7.3 (5.0) in the Study and 5.3 (2.9) in the Control cycle (P = 0.018). Although the duration of stimulation seemed longer in the Study compared to Control cycle, 10.1 (1.5) and 8.9 (2.7) days respectively (P = 0.05), the same trigger criterion was defined and used in the two cycles confirmed by a similar mean diameter of the two biggest follicles on trigger day: 18.5 (1.1) vs. 18.3 (1.1) mm in the Study and Control cycle, respectively (P = 0.91). The sample size calculation was based on the follicular output rate, and the study was not powered to look at oocyte retrieval rates. Bigger studies are needed to confirm this finding. Although no increase in the follicular output rate was demonstrated, significantly more oocytes were retrieved after eight weeks of hCG priming, which is an important finding in a population of women with low ovarian reserve. Benefits of long term low dose hCG priming must be confirmed in a larger study. NCT04643925