Eight weeks of androgen priming by daily low-dose hCG injections before ICSI treatment in women with low ovarian reserve.

STUDY QUESTION: Does 8 weeks of continuous low-dose hCG administration increase the proportion of antral follicles that reach the preovulatory state during ovarian stimulation (OS) in women with low ovarian reserve? SUMMARY ANSWER: The proportion of antral follicles (2-10 mm) that reached the preovulatory state did not increase. WHAT IS KNOWN ALREADY: The administration of androgens prior to OS might upregulate FSH receptor (FSHR) expression on granulosa cells, making follicles more responsive to exogenous FSH stimulation during OS. LH and hCG stimulate the local follicular androgen synthesis in theca cells and may be used as an endogenous androgen priming method. Exogenous priming by testosterone and dehydroepiandrosterone (DHEA) have been shown to increase the number of retrieved oocytes and live birth rate but the studies are small, and their use is associated with side effects. STUDY DESIGN, SIZE, DURATION: A prospective, paired, non-blinded single-center study including 20 women serving as their own controls conducted between January 2021 and July 2021 at The University Hospital Copenhagen Rigshospitalet, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants underwent two identical consecutive IVF/ICSI treatments, a Control cycle and a Study cycle, separated by ∼8 weeks (two menstrual cycles) of daily injections of 260 IU recombinant hCG (rhCG). A freeze-all strategy was applied in the Control cycle. Both IVF/ICSI cycles were performed in a fixed GnRH antagonist protocol using a daily dose of 300 IU recombinant FSH (rFSH) and GnRH antagonist 0.25 mg from stimulation days 5-6. MAIN RESULTS AND THE ROLE OF CHANCE: Follicular output rate, defined as the number of follicles >16 mm on hCG trigger day divided by the antral follicle count (2-10 mm) at baseline, did not increase after 8 weeks of hCG priming (P = 0.8). The mean number of oocytes retrieved was significantly higher after the hCG priming being 4.7 (2.8) vs 3.2 (1.7) in the Study and Control cycle, respectively (P = 0.01). The duration of stimulation was longer in the Study versus the Control cycle (P = 0.05), despite the use of identical hCG trigger criterion and similar diameters of the three biggest follicles on hCG trigger day in the two cycles (P = 0.9). LIMITATIONS, REASONS FOR CAUTION: The sample size was small, and the number of oocytes retrieved was not the primary endpoint. Larger studies are needed to confirm this finding. WIDER IMPLICATIONS OF THE FINDINGS: Long-term, low-dose rhCG administration may increase the number of oocytes retrieved during IVF/ICSI in women with low ovarian reserve, but more research is needed before firm conclusions can be drawn. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by an unrestricted grant from Gedeon Richter. A.P. reports personal consulting fees from PregLem SA, Novo Nordisk A/S, Ferring Pharmaceuticals A/S, Gedeon Richter Nordics AB, Cryos International, and Merck A/S outside the submitted work and payment or honoraria for lectures from Gedeon Richter Nordics AB, Ferring Pharmaceuticals A/S, Merck A/S, and Theramex and Organon & Co. Grants to the institution have been provided by Gedeon Richter Nordics AB, Ferring Pharmaceuticals A/S, and Merck A/S and receipt of equipment by the institution from Gedeon Richter Nordics AB is reported. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT04643925.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-ß family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.

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

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.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold.

STUDY QUESTION: Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? SUMMARY ANSWER: We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. WHAT IS KNOWN ALREADY: Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. STUDY DESIGN, SIZE, DURATION: Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N = 241) and human (N = 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Incubation in 0.1% SDS for 18-24 hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. STUDY FUNDING/COMPETING INTEREST(S): This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

A simple method to quantify follicle survival in cryopreserved human ovarian tissue.

STUDY QUESTION: Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically? SUMMARY ANSWER: A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluation and evidence for a consistently high follicle survival in a series of ovarian tissue from 25 Danish girls and women undergoing ovarian tissue cryopreservation (OTC) was obtained. WHAT IS KNOWN ALREADY: Securing follicle survival in cryopreserved ovarian tissue is crucial for proper quality control when centers wish to implement OTC. The only established technique for validation of follicle survival is xenografting of thawed ovarian tissue to immunodeficient mice. However, this functional test is expensive, time consuming, requires animal facilities and only provides a qualitative-not quantitative-measure for follicle survival. STUDY DESIGN SIZE, DURATION: Quantification of follicle survival in human ovarian tissue donated from 30 girls and women having tissue cryopreserved for fertility preservation from 2000 to 2015 at the Laboratory of Reproductive Biology in Copenhagen, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian cortex was donated from 25 girls and young women aged 10-36 years (mean age: 25 years) and the average storage time in liquid nitrogen was 9.1 ± 5.6 years, ranging from 1.6 to 17.9 years. In 12 of the cases, the ovarian tissue was collected from the local hospital and in the other 13 cases the ovarian tissue was transported on ice up to 6 h prior to freezing. Donated fresh ovarian surplus tissue was obtained from five women aged 23-34 years (mean age: 27 years). Ovarian tissues were chopped into small fragments and incubated in culture medium containing 50 mg/ml NR for 3-4 h. Fragments of ovarian tissue containing clearly NR-stained follicles were selected for counting, encapsulated in 4% agar and were processed for histology to calculate a follicular survival rate. MAIN RESULTS AND THE ROLE OF CHANCE: The mean follicle survival rate in the 25 patients after freezing and thawing was 84% ± 11 (mean ±SD), ranging from 50% to 98%. The high follicle survival rate in this clinical series of patients reflects a constant high-quality service performed in our center and confirms the robustness of the slow freezing protocol. No significant association between follicle survival rates and storage time was found using linear regression analysis, suggesting that storage in liquid nitrogen does not affect viability of the tissue. No significant association in follicle survival rates was found between ovarian tissues collected at the local hospital compared to tissues transported on ice prior to freezing, supporting that prolonged cooling does not seem to greatly affect the follicle survival. For the fresh ovarian tissue, the average follicle survival rate was 91% ± 5 (mean ± SD) in five patients, ranging from 81% to 95%. LIMITATIONS, REASONS FOR CAUTION: Even though the NR staining requires active incorporation of the dye, the test is merely a short in situ test that cannot completely replace the functional value of xenografting studies in which the integrity and developmental potential of the ovarian follicles are assessed. WIDER IMPLICATIONS OF THE FINDINGS: OTC is now being employed around the world but to date it has been difficult for centers to evaluate the effectiveness of their program and perform proper quality control. NR staining combined with histological evaluation is the first quantitative method to provide a survival rate for follicles in frozen-thawed human ovarian tissue and offer a valuable and easily applicable tool to validate the cryopreservation procedure when implementing OTC or as routine quality control for the overall freezing performance within tissue banking facilities. STUDY FUNDING/COMPETING INTEREST(S): The Research Pools of Rigshospitalet, the Danish Cancer Foundation, Dagmar Marshalls Foundation, and the Novo Nordic Foundation are thanked for having funded this study. The authors have no conflicts of interest.

Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort.

PURPOSE: The purpose of the study is to review all peer-reviewed published reports of women receiving ovarian tissue transplantation (OTT) with frozen/thawed tissue (OTC) with respect to age, diagnosis, transplantation site, fertility outcome, and potential side effects, including data from all women in the Danish program. METHODS: A systematic review of the literature was performed in PubMed combined with results from all patients who had received OTT in Denmark up to December 2017. RESULTS: OTT has been reported from 21 different countries comprising a total of 360 OTT procedures in 318 women. In nine women, malignancy was diagnosed after OTT; none were considered to be directly caused by the OTT. Despite a potential under reporting of cancer recurrence, there is currently no evidence to suggest that OTT causes reseeding of the original cancer. Renewed ovarian endocrine function was reported in 95% of the women. Half of all children born following OTT resulted from natural conception, and newborns were reported to be healthy except for one neonate with a chromosome anomaly with a family disposition. Women who conceived after OTT were significantly younger than those who failed. CONCLUSION: This study found no indications of sufficient numbers of malignant cells present in the ovarian tissue to cause recurrence of cancer after OTT. Further, it is unlikely that OTC affects the well-being of children born. OTC is now an established method of fertility preservation in Denmark with public reimbursement. The current data encourage that women who require gonadotoxic treatment should be offered an individual evaluation considering fertility preservation.

Pathology and localization of Avibacterium endocarditidis in experimentally infected broiler breeders.

The aim of this study was to investigate the pathogenic potential of Avibacterium endocarditidis. Forty broiler breeders were inoculated with one of three doses of the organism and killed at different time points. Bacteriology, pathology and fluorescence in-situ hybridization (FISH) were performed to evaluate bacterial growth and the development of lesions. Abundant growth in pure culture of A. endocarditidis was always obtained from valvular lesions, while only poor growth or no growth was obtained from liver and spleen lesions, confirming previous observations from naturally occurring cases. Gross lesions and histopathological findings confirmed previous observations. Valvular lesions were acute, ranging from slight thickening of the valves to severe inflammation. In most cases, bacteria colonized the valves. Lesions observed in the spleen included different degrees of necrosis and liver lesions ranged from very small infarcts to large areas of coagulative necrosis. Arthritis occurred in 19 birds, 15 of which tested positive for A. endocarditidis. Most birds developed bacteraemia, but the inability to isolate bacteria from the liver and spleen and the lack of bacteria demonstrated by FISH and histopathology, suggested lack of fulminant septicaemia. A significant correlation between the size of dose inoculated and development of valvular endocarditis was not observed; however, regressive changes in the ovary, liver necrosis and hepato-, spleno- and renomegaly were significantly dose dependent. A. endocarditidis represents a potential pathogen for chickens, the reservoir of which remains to be determined.

Pathology, tissue metalloproteinase transcription and haptoglobin responses in mice after experimental challenge with different isolates of Pasteurella multocida obtained from cases of porcine pneumonia.

Pasteurella multocida is a major cause of porcine pneumonia, but the pathogenesis of the disease is poorly defined. The aim of this study was to further understand the host response to infection by use of a mouse model of P. multocida pneumonia. Twenty female mice were divided into four groups (n=5). Three groups were infected with one of three isolates of P. multocida isolated from clinical cases of chronic porcine pneumonia with necrotizing, suppurative and non-suppurative lesions, respectively. The fourth group served as uninfected controls. Mice were killed 24 h postinfection and samples were collected for bacteriology, histopathology and in-situ hybridization for detection of P. multocida. Measurements of expression of genes encoding matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) in lung tissue and quantification of serum haptoglobin concentration were performed. P. multocida was found in the lung and spleen. Lung lesions were characterized by deposition of fibrin in alveoli and bronchioles, perivascular oedema, suppuration and necrosis. The cellular infiltration was mainly of neutrophils. Splenic neutrophilic infiltration was also evident. Minor differences in the severity and nature of lesions were seen according to the isolate of P. multocida used for infection. Intranasal infection of mice can therefore be used to evaluate the host response and lesions caused by P. multocida obtained from porcine pneumonic infections. The inflammatory response in this model is associated with increased tissue expression of genes encoding MMP9, TIMP1 and serum haptoglobin concentration.

Occurrence and tissue distribution of porcine circovirus type 2 identified by immunohistochemistry in Danish finishing pigs at slaughter.

Infection with porcine circovirus type 2 (PCV2) may be subclinical or lead to the development of porcine circovirus disease (PCVD), which includes the entities of post-weaning multisystemic wasting syndrome (PMWS) and the porcine respiratory disease complex (PRDC). PCV2 infection and PMWS occur in the early post-weaning period and are also recognized in finishing pigs of 12-19 weeks of age. The aim of the present study was to assess the role of PCV2 infection in disease of finishing pigs. Accordingly, the occurrence and tissue distribution of PCV2 was examined in Danish finishing pigs at the time of slaughter. Multiple lymph nodes and the spleen, lungs and kidneys from 136 pigs with PRDC (case group) and 36 pigs without lung lesions (control group) were examined by immunolabelling for the presence of PCV2. Additionally, follicular dendritic cells (FDC) were identified immunohistochemically. One or more tissues of 61% of the pigs were positive for PCV2 antigen. Up to 78% of the pigs had mild lymphoid depletion, indistinct lymphoid follicles and/or histiocytic infiltration of the lymph nodes, but these lesions were not associated with PCV2. No association was found between the presence of lung or kidney lesions and detection of PCV2. Three distinct patterns of cellular PCV2 antigen labelling were recognized: (1) labelling of cells with stellate morphology and reticular distribution, (2) labelling of isolated non-epithelial, cells, and (3) epithelial labelling. The reticular pattern was most common and localized to the centres of lymphoid follicles, corresponding to the morphology and distribution of FDCs. This observation may be interpreted to suggest that PCV2 may interact with FDCs to cause depletion of B lymphocytes. Alternatively, the FDCs may be a reservoir of infective PCV2 in subclinically infected animals or represent a simple storage site for PCV2 antigen in pigs that have recovered from infection.

An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark.

Respiratory infections are among the most important diseases of growing pigs. In order to elucidate the multifactorial aetiology of porcine respiratory disease complex (PRDC) in Denmark, lungs from 148 finishing pigs with cranioventral bronchopneumonia (case group) and 60 pigs without lung lesions (control group) were collected from abattoirs. The pathogens involved in PRDC and their interactions were identified and linked to the histopathological diagnosis. The lung samples were cultured for bacteria and tested by multiplex polymerase chain reaction for presence of swine influenza virus (type A), porcine reproductive and respiratory syndrome virus (both European and US type), porcine circovirus type 2 (PCV2), porcine respiratory coronavirus, porcine cytomegalovirus, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. All cases had cranioventral lobular bronchopneumonia consistent with PRDC. There was a broad range of microscopical lesions and the cases were characterized as acute (n=10), subacute (n=24) or chronic (n=114) bronchopneumonia. Five bacterial species, five viruses and two Mycoplasma spp. were detected in different combinations. PCV2, M. hyopneumoniae, M. hyorhinis and Pasteurella multocida were detected most frequently among the PRDC affected swine and the diversity and number of pathogens were higher in these animals compared with controls. No clear-cut associations were detected between pathogens and histological lesions or histopathological diagnoses. PRDC occurs more frequently than enzootic pneumonia among Danish finishing pigs and has complex and varied histopathology.