Midkine characterization in human ovaries: potential new variants in follicles.

OBJECTIVE: To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. DESIGN: Descriptive study. SETTING: University Hospital. PATIENTS: The study included samples from 121 patients: 71 patients (aged 17-37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20-35 years) receiving in vitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. INTERVENTIONS: None. MAIN OUTCOME MEASURES: MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. RESULTS: Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in SAF compared with preovulatory follicles. In addition, different molecular weight species of MDK were detected using western blotting in various ovarian sample types: GC and FF samples presented primarily one band of approximately 15 kDa and an additional band of approximately 13 kDa, although other bands with higher molecular weight (between 30 and 38 kDa) were detected in medulla tissue. CONCLUSIONS: This is the first time that MDK has been immunolocalized in human ovarian cells at the protein level and that potentially different MDK variants have been detected in human FF, GC, and ovarian medulla tissue. Future studies are needed to sequence and identify the different potential MDK variants found to determine their functional importance for ovary and oocyte competence.

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100-130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E2), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E2 and FSH to the culture medium.

Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue.

BACKGROUND: Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. METHODS: Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24-36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150-200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. RESULTS: A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). CONCLUSIONS: Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.

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

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.

Organotypic Culture of Testicular Tissue from Infant Boys with Cryptorchidism.

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.

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

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.

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

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 ±â€…189 pg/mL [mean ±â€…SEM], n = 188; non-PCO: 3498 ±â€…199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ±â€…40 pg/mL, n = 125; non-PCO: 573 ±â€…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.

Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation.

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.

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

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.

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

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.

Histidine buffered media maintains pH stabile during cooled transportation of human ovarian tissue.

The aim of this study was to investigate whether pH is stable when transporting ovarian tissue in media buffered with either HEPES or histidine. Furthermore, if the choice of transport media impacts the in vitro maturation rate of oocytes collected in connection with ovarian tissue cryopreservation. Human ovaries (n = 34) collected for ovarian tissue cryopreservation were transported immersed in either 30 ml of HEPES buffered (follicle flushing media (Origio; Denmark)) or histidine buffered media (Custodiol®-HTK, Koehler-Chemie, Germany). Tissue was transported on ice for 4-5 h. At arrival, the ovary was weighed, and the pH of the media was measured at 0 °C. From 15 patients, immature oocytes were collected for in vitro maturation, oocytes that matured to metaphase II were evaluated. The pH measured in the HEPES buffered media (pH = 7.5 ± 0.13, n = 18) was significantly higher (p < 0.001) than the pH measured in the histidine buffered media (pH = 7.2 ± 0.05, n = 16). The standard deviation of pH measurements for the histidine buffered media was significantly lower than for the HEPES buffered media measurements (p < 0.0001). A total of 170 and 247 immature oocytes were collected and in vitro matured from ovaries transported in HEPES and histidine buffered media, respectively. The maturation rate of immature oocytes after IVM was similar in the two groups. The results show that pH in the histidine buffered media is closer to the physiological level and more stable than in HEPES buffered medium and support the use of histidine buffered media for cooled transportation of human ovaries.

Effects of Er:YAG laser treatment on re-vascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice.

PURPOSE: The huge loss of ovarian follicles after transplantation of frozen/thawed ovarian tissue is considered a major drawback on the efficacy of the procedure. Here we investigate whether Er:YAG laser treatment prior to xenotransplantation can improve re-vascularization and subsequently follicle survival in human ovarian tissue. METHODS: A total of 99 frozen/thawed human ovarian cortex pieces were included of which 72 pieces from 12 woman were transplanted to immunodeficient mice. Tissues from each woman were included in both an 8-day and an 8-week duration study and treated with either full-beam laser (L1) or fractionated laser (L2), or served as untreated controls. Vascularization of the ovarian xenografts were evaluated after 8 days by qPCR and murine Cd31 immunohistochemical analysis. Follicle densities were evaluated histologically 8 weeks after xenografting. RESULTS: Gene expression of Vegf/VEGF was upregulated after L1 treatment (p=0.002, p=0.07, respectively), whereas Angpt1, Angpt2, Tnf-α, and Il1-ß were significantly downregulated. No change in gene expression was found in Cd31/CD31, ANGPT1, ANGPT2, ANGTPL4, XBP1, or LRG1 after any of the laser treatments. The fraction of Cd31 positive cells were significantly reduced after L1 and L2 treatment (p<0.0001; p=0.0003, respectively), compared to controls. An overall negative effect of laser treatment was detected on follicle density (p=0.03). CONCLUSIONS: Er:YAG laser treatment did not improve re-vascularization or follicle survival in human ovarian xenografts after 8 days and 8 weeks grafting, respectively. However, further studies are needed to fully explore the potential angiogenic effects of controlled tissue damage using different intensities or lasers.

Use of cryopreserved ovarian tissue in the Danish fertility preservation cohort.

OBJECTIVE: To evaluate the use of cryopreserved ovarian tissue in the Danish fertility preservation cohort. DESIGN: Retrospective cohort study. SETTING: University hospitals and fertility clinics. PATIENT(S): Ovarian tissue cryopreservation (OTC) was performed for 1,186 Danish girls and women from 1999-2020, of whom 117 subsequently underwent ovarian tissue transplantation (OTT). Subgroup 1 included 759 patients with a follow-up period of >5 years. Out of these, OTT rates were further analyzed for those patients who were alive and aged >24 years in July 2020 (subgroup 2; n = 554). INTERVENTION(S): OTC and OTT. MAIN OUTCOME MEASURE(S): OTT, death, donation of tissue. RESULT(S): In subgroup 1, 14% of the patients had undergone OTT, 18% had died, 9% had donated their tissue for research, and 59% still had their tissue stored. In subgroup 2, 19% had undergone OTT and for most diagnoses the OTT rates ranged from 15% to 22% with benign hematologic diseases having the highest OTT rate (35%). On the basis of the entire cohort, stratified age analysis indicated that women aged ≥30 years at OTC were more likely to return for OTT than women aged 18-29 years at OTC; mean storage times were 3.7 and 3.6 years, respectively. Only 4% of the girls aged <18 years at OTC had undergone OTT. CONCLUSION(S): The OTT rates depended on the diagnosis, age at OTC, and follow-up time. Specific criteria are needed for reporting and comparing OTT rates. Six out of 10 patients still had their cryopreserved tissue stored and longer follow-up is needed, especially for younger girls.

A threshold concentration of FSH is needed during IVM of ex vivo collected human oocytes.

PURPOSE: To investigate the effect of different FSH concentrations on human oocyte maturation in vitro and its impact on gene expression of key factors in the surrounding cumulus cells. METHODS: The study included 32 patients who underwent unilateral oophorectomy for ovarian tissue cryopreservation (OTC) (aged 28 years on average). Immature oocytes were collected from surplus medulla tissue. A total of 587 immature oocytes were divided into three categories according to the size of the cumulus mass: large (L-COCs), small (S-COCs), and naked oocytes (NOs), and submitted to 44-h IVM with one of the following concentrations of recombinant FSH: 0 IU/L, 20 IU/L, 40 IU/L, 70 IU/L, or 250 IU/L. After IVM, oocyte nuclear maturation stage and diameter were recorded. The relative gene expression of FSHR, LHCGR, and CYP19A1 in cumulus cells before (day 0; D0) and after IVM were evaluated. RESULTS: Addition of 70 or 250 IU/L FSH to the IVM medium improved oocyte nuclear maturation compared to 0, 20, and 40 IU/L FSH by upregulating LHCGR and downregulating FSHR in the cumulus cells. CONCLUSION: FSH improved oocyte nuclear maturation at concentrations above 70 IU/L suggesting a threshold for FSH during IVM of ex vivo collected human oocytes from small antral follicles. Moreover, current results for the first time highlight that FSH function in vitro is mediated via cumulus cells by downregulating FSHR and upregulating LHCGR, which was also observed when the immature oocytes progressed in meiosis from the GV to the MII stage.

Clusters of smooth endoplasmic reticulum are absent in oocytes from unstimulated women.

RESEARCH QUESTION: What is the frequency of morphological dysmorphisms in immature human oocytes collected ex vivo from small antral follicles and matured in vitro? DESIGN: Human ovaries (n = 56) were excised for ovarian tissue cryopreservation (OTC). None of the patients had received exogenous gonadotrophins prior to the procedure. Immature oocytes released from small antral follicles were collected in connection with isolation of the cortex for OTC. The oocytes' maturation stage and the morphological characteristics of the cytoplasm, zona pellucida, perivitelline space and first polar body were assessed after in-vitro maturation (IVM). RESULTS: A total of 1649 immature oocytes were collected: 30% of oocytes matured to the metaphase II (MII) stage after IVM, while metaphase I (MI), germinal vesicle and degenerated oocytes accounted for 20%, 24% and 26%, respectively. The percentages of oocytes without any dysmorphisms were 53%, 92%, and 97% for the MII, MI and germinal vesicle stage oocytes, respectively. The most frequently observed dysmorphisms among the MII oocytes were first polar body fragmentation (22%), homogeneously distributed cytoplasmic granularity (16%) and an enlarged perivitelline space (14%). Interestingly, none of the oocytes at any stage had clusters of smooth endoplasmic reticulum (SER). CONCLUSIONS: Morphological dysmorphisms are present among in-vitro-matured oocytes at all maturation stages. The incidence of dysmorphisms increases as maturation progresses. The most frequent dysmorphism among MII oocytes after IVM was fragmentation of the first polar body. Clusters of SER were not observed in oocytes from unstimulated patients.

High Variability of Molecular Isoforms of AMH in Follicular Fluid and Granulosa Cells From Human Small Antral Follicles.

Anti-Müllerian hormone (AMH) is a member of the TGF-ß superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive life. AMH is thought to inhibit aromatase (i.e., CYP19) expression and decrease the conversion of androgens to oestrogens, especially in small antral follicles before dominance is achieved. Thus, AMH acts as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH has not been fully elucidated. The present study measured and determined AMH isoforms in human follicular fluid (FF) from small antral follicles and in human GCs using four ELISAs, western blot, and immunofluorescence analysis. We evaluated the presence of the following isoforms: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. A negative correlation between follicle diameter and the AMH forms was detected. Moreover, western blot analysis detected various AMH forms in both FFs and GCs, which did not match our consensus forms, suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size in the same woman. This study detected several AMH forms in FF and GCs obtained from human small antral follicles, which suggests that intrafollicular processing of AMH is complex and variable. Thus, it may be difficult to develop an antibody-based AMH assay that detects all AMH isoforms. Furthermore, the variability between follicles suggests that designing a recombinant AMH standard will be difficult.

N-acetylcysteine protects ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue.

STUDY QUESTION: Can antioxidant treatment with N-acetylcysteine (NAC) protect ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue? SUMMARY ANSWER: Daily administration of NAC for 7-12 days post-transplantation reduced ischemia-reperfusion injury and increased follicle survival in human ovarian xenografts by upregulating the antioxidant defense system and exerting anti-inflammatory and antiapoptotic effects. WHAT IS KNOWN ALREADY: Freezing of human ovarian tissue is performed with high follicular survival rates but up to 70% of follicles appear to be lost due to hypoxia and ischemia-reperfusion injury during ovarian tissue transplantation (OTT). NAC has been demonstrated to possess antioxidant and antiapoptotic properties, and studies in rodents have shown that intraperitoneal administration of NAC reduces ischemia-reperfusion injury and increases follicle survival in autotransplanted murine ovaries. STUDY DESIGN, SIZE, DURATION: Pieces of frozen-thawed human ovarian tissue from 28 women aged 23-36 years were transplanted to immunodeficient mice in short- and long-term xenograft studies or cultured in vitro. Three short-term xenograft studies (1-week duration) were performed, in which saline or 150 mg/kg NAC was administered for 7 days post-transplantation (n = 12 patients per group). Two long-term xenograft studies (4 weeks of duration) were performed. In one of these studies, saline or 150 mg/kg NAC was administered for 12 days (n = 12 patients per group), while in the other study 50, 150 or 300 mg/kg NAC was administered for 7 days (n = 8 patients per group). In addition, human ovarian tissue (n = 12 pieces from three patients per group) was cultured with increasing concentrations of NAC (0, 5, 25 and 75 mM) for 4 days in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated ovarian tissue was obtained from women who had undergone ovarian tissue cryopreservation for fertility preservation at the University Hospital of Copenhagen. Cortical tissue pieces (5 × 5 × 1 mm) were transplanted subcutaneously to immunodeficient mice and NAC or saline was injected intraperitoneally. Grafts were retrieved after 1 or 4 weeks and follicle density was assessed. Gene expression analysis of antioxidant defense markers (superoxide dismutase; Sod1/SOD1, heme oxygenase-1; Hmox1/HMOX1, catalase; Cat/CAT), proinflammatory cytokines (tumor necrosis factor-alpha; Tnf-α, interleukin-1-beta; Il1-ß, interleukin 6; Il6), apoptotic factors (B-cell lymphoma 2; Bcl2/BCL2, Bcl-2-associated X protein; Bax/BAX) and angiogenic factors (vascular endothelial growth factor A; Vegfa/VEGFA, angiopoietin-like 4; Angptl4/ANGPTL4) was performed in 1-week-old human ovarian xenografts and in cultured human ovarian tissue. Grafts retrieved after 4 weeks were histologically processed and analyzed for vascularization by CD31 immunohistochemical staining, fibrosis by Masson's Trichrome staining and apoptosis by immunofluorescence using cleaved caspase-3. MAIN RESULTS AND THE ROLE OF CHANCE: After 1-week grafting, the relative expression of Sod1, Hmox1 and Cat was significantly higher in the group receiving 150 mg/kg NAC (NAC150-treated group) compared to controls (P = 0.04, P = 0.03, and P = 0.01, respectively), whereas the expression levels of Tnf-α, Il1-ß and Il6 were reduced. The Bax/Bcl2 ratio was also significantly reduced in the NAC150-treated group (P < 0.005). In vitro, the relative gene expression of SOD1, HMOX1 and CAT increased significantly in the human ovarian tissue with increasing concentrations of NAC (P < 0.001 for all genes). However, the expression of VEGFA and ANGPTL4 as well as the BAX/BCL2 ratio decreased significantly with increasing concentrations of NAC (P < 0.02, P < 0.001 and P < 0.001, respectively). After 4-week grafting, fibrosis measured by collagen content was similar in the NAC150-treated group compared to controls (control: 56.6% ± 2.2; NAC150: 57.6% ± 1.8), whereas a statistically significant reduction in the CD31-positive vessel area was found (control: 0.69% ± 0.08; NAC150: 0.51% ± 0.07; P < 0.02). Furthermore, a reduced immunoreactivity of cleaved caspase-3 was observed in follicles of the NAC150-treated xenografts compared to controls. Follicle density (follicles/mm3, mean ± SD) was higher in the NAC150-treated group compared to the control group in the 1-week xenografts (control: 19.5 ± 26.3; NAC150: 34.2 ± 53.5) and 4-week xenografts (control: 9.3 ± 11.0; NAC150: 14.4 ± 15.0). Overall, a 2-fold increase in follicle density was observed in the NAC150-group after 1-week grafting where fold changes in follicle density were calculated in relation to grafts from the same patient. Around a 5-fold increase in follicle density was observed in the NAC150 and NAC300 groups after 4-week grafting. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Follicle density in the human ovarian cortex is highly heterogeneous and can vary 100-fold between cortex pieces from the same woman. 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. While intraperitoneal injections of NAC appeared to reduce ischemia-reperfusion injury in human ovarian xenografts, different administration routes should be investigated in order to optimize NAC for potential clinical use. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to demonstrate the antioxidant, anti-inflammatory and antiapoptotic properties of NAC in xenotransplanted human ovarian tissue. Therefore, NAC appears to be a promising candidate for protecting ovarian follicles from ischemia-reperfusion injury. This provides the initial steps toward clinical application of NAC, which could potentially reduce the loss of ovarian follicles following OTT. STUDY FUNDING/COMPETING INTEREST(S): We are grateful to the Danish Childhood Cancer Foundation, Hørslev Foundation, Aase and Einar Danielsen's Foundation (grant number: 10-001999), Dagmar Marshalls Foundation, Else and Mogens Wedell-Wedellsborgs Foundation, Knud and Edith Eriksens Mindefond, and Fabrikant Einar Willumsens Mindelegat for funding this study. None of the authors have any competing interests to declare.

Proteome of fluid from human ovarian small antral follicles reveals insights in folliculogenesis and oocyte maturation.

STUDY QUESTION: Is it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)? SUMMARY ANSWER: The largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes. WHAT IS KNOWN ALREADY: Protein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis. STUDY DESIGN, SIZE, DURATION: Proteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation. LIMITATIONS, REASONS FOR CAUTION: A possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons. WIDER IMPLICATIONS OF THE FINDINGS: This study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation. STUDY FUNDING/COMPETING INTEREST(S): The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors. TRIAL REGISTRATION NUMBER: N/A.

Comparison and assessment of necropsy lesions in end-of-lay laying hens from different housing systems in Denmark.

Apperantly healthy laying hens at the end of production (60 to 91 wk) were investigated for the occurrence of pathology and bacterial infections. In total, 7,477 hens from 15 flocks representing the following production systems: Enriched cages, barn housed layers, and organic/free range layers were necropsied. Indications of bacterial infection were investigated by bacteriological cultivation. The overall prevalence of lesions was 16.60%, including lesions of both infectious and non-infectious origin. The most prevalent lesions were bursitis presternalis (6.65%), reproductive tract lesions (e.g., salpingitis and/or peritonitis and/or oophoritis) (3.50%), serosal scarification (e.g., fibrotic adhesive peritonitis) 1.55%, and neoplasm 1.73%. Significant differences were observed between different production systems and/or flocks in the prevalence of reproductive tract lesions, bursitis presternalis, serosal scarification, skin infections, juvenile hens, and traumas/fractures. No significant difference was observed between different production systems in the prevalence of neoplasia, infection of septicemic etiology, and pododermatitis. In total, 3.4% of the hens were out of lay, with significantly higher rate in organic flocks. Infections of the reproductive tract were the most prevalent lesions with bacterial etiology in all productions systems. In total, 40% of the hens with lesions associated to the oviduct were out of lay and significant difference between production systems were observed. Escherichia coli was the most commonly isolated bacteria and in 90% of the cases they were isolated from the reproductive tract lesions. The second most prevalent bacteria was Gallibacteruim anatis. Significant difference in the prevalence of E. coli positive hens was observed between production systems (P < 0.05). In conclusion, the prevalence of reproductive tract lesions in apparently healthy end-of-lay laying was higher than indicated in previous reports. These findings support the previous suggestions that E. coli and G. anatis are the major pathogens causing reproductive tract lesions.

Effect of whey protein supplementation on sperm quality and fertility in male mice.

Protein supplements are a billion-dollar industry and the intake of these supplements is increasing, especially among young men. However, little is known about whether consumption of these products affects the reproductive health. The aim of this study was to assess the effect of whey protein supplementation on the sperm quality and reproductive health of male mice. A total of 48 male NMRI mice were fed with either plain tap water or a high dose of whey protein (Whey100, BodyLab) supplemented in the drinking water for 3 months. Mice was individually housed with two female mice for five days and reproductive parameters were assessed. DNA fragmentation index (DFI) was assessed at 0 h and 4 h of in vitro incubation using a sperm DNA integrity test (SDI®-test). No significant differences were detected between the groups in the epididymal sperm count, sperm motility, DFI, oxidation-reduction potential (ORP), serum testosterone, body and seminal vesicles weights, relative testis and epididymal weights, testicular morphology, number of impregnated females, or litter size. No correlation was found between ORP and DFI. These results suggest that the highest recommended human dose of whey protein supplementation do not significantly impair the sperm quality and fertility in male mice.