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Amy Miller, Elainna Jentz, and Cassandra Duncan

Graphical abstract

13-lined ground squirrels (TLGS; Ictidomys tridecemlineatus) are small, omnivorous, fossorial, hibernating sciurids. TLGS are seasonal induced ovulators, with a ~28-day gestation period. The main goal of this study was to ascertain whether enzyme-linked immunosorbent assay (ELISA) of TLGS fecal samples can be used to non-invasively detect pregnancy. Competitive ELISAs for progestogen metabolites were conducted on feces collected from a group of (n =13) females. Feces were collected thrice weekly during the breeding season and frozen for subsequent analysis. Competitive ELISAs were run using progesterone kits ), setting data against seven different time-points between hibernation, emergence, and litter birthdate. Eleven females produced litters. ELISA data from the (n = 2) non-pregnant females demonstrated no rise in progestogen metabolites at any point over 28 days. In contrast, data from the (n = 11) pregnant females all demonstrated a pronounced rise in progestogen metabolites, with most animals displaying progesterone withdrawal in the final week of gestation. A >20-fold rise in progestogen metabolite was observed halfway through gestation (P < 005). Analysis on litter size and progestogen metabolite concentration showed no significant correlation (r2 = −0.615). Initial correlation analysis done on sex ratio of litters vs progestogen metabolites showed no significant effect of progesterone on sex ratios (males: r2 = −0.772, females: r2 = 0.375). This work demonstrated that TLGS also undergo progesterone withdrawal about a week before parturition. We have ascertained that a commercially available progesterone assay kit can detect a significant elevation in progestogen metabolites in this species about halfway through gestation.

Lay summary

This research was conducted to discover whether pregnancy prediction is possible in female 13-lined ground squirrels (TLGS; a small hibernating ground squirrel named for their number of stripes). Pregnancy status in this species, we postulated, could be anticipated by generating profiles for individuals via a non-invasive technique known as fecal endocrine hormone profiling. Fecal samples were collected from 13 females thrice weekly for 4 weeks post-hibernation in the breeding season of 2016. Fecal samples were then processed and run through an assay known as an ELISA giving concentrations of hormone metabolites excreted through feces. We then set these samples against time points to develop a profile for each female. We have ascertained that elevated progesterone (potential pregnancy) can be detected by a commercially available assay kit. Understanding hormone patterns in animals gives researchers a better idea of best husbandry practices, including breeding in managed care.

Daniella Gilboa, Liron Seidman, Polina Kimiagarov, Avia Noni, Ravid Doron, and Daniel S Seidman

Objective

Oocyte pick-up (OPU) is a painful but essential part of in-vitro fertilization (IVF) that is usually performed under sedation and analgesia (SaA). Our aim was to study that why some women decide to undergo OPU without SaA?

Methods

This was a prospective study using patient questionnaires and the standardized 7-item generalized anxiety disorder (GAD-7) score. The patients were asked to assess the pain experienced during OPU using a visual analog scale (VAS). The study sample was a convenience sample of 100 healthy women undergoing OPU at our unit with or without SaA.

Results

Women who chose to undergo OPU without SaA were significantly more likely to express the fear of anesthesia. A high pain score (VAS ≥ 6) was reported by significantly more patients who underwent OPU without SaA than with SaA. Yet, 98% of the patients who underwent OPU without SaA stated that in future IVF cycles, they would still choose to undergo OPU without SaA. More patients had high anxiety scores among those who underwent OPU with than without SaA.

Conclusions

Women who chose to undergo OPU without SaA reported more often fear of anesthesia. Although these women experienced significantly more pain during OPU, almost all of them suggested that they would still choose to undergo OPU without SaA. Increased anxiety, as expressed by higher GAD-7 scores, was not associated with a tendency to choose SaA during OPU. The option of OPU without SaA seems to be an acceptable option for selected women.

Lay summary

Egg retrieval from the ovaries is a painful part of in vitro fertilization (IVF). It is, therefore, usually performed under sedation and pain relief (analgesia). The aim of this study was to investigate: Why some women decide to undergo egg retrieval without sedation? We prospectively studied 100 women using patient questionnaires and standardized scores in order to measure patient's pain and anxiety levels. We found that women who chose to undergo egg retrieval without sedation were significantly more likely to express fear of anesthesia. As expected, women who decided to forgo sedation experienced more pain during egg retrieval, yet, 98% of them decided that in future IVF cycles, they would still choose to undergo egg retrieval without sedation. Surprisingly, women who had high anxiety scores were not more likely to ask for sedation during egg retrieval. The option to undergo egg retrieval without sedation during IVF seems to be acceptable for some women.

Camila N Ortiz, Annelyn Torres-Reverón, and Caroline B Appleyard

Endometriosis is a complex disease characterized by inflammation and the growth of endometrial- like glands and stroma outside the uterine cavity. The pathophysiology of endometriosis is not entirely understood, however, with a prevalence of ~10% of women in their reproductive years, the disease symptoms significantly affect the quality of life of millions of women globally. Metabolomic studies have previously identified specific metabolites that could be a signature of endometriosis. This approach could potentially be used as a non-invasive tool for early diagnosis and provide a better understanding of endometriosis pathophysiology. This review aims to provide insight as to how endometriosis affects the metabolome by reviewing different studies that have used this approach to design follow-up studies. The search query included the term 'endometriosis' in combination with 'metabolomics', 'lipidomics', or 'sphingolipidomics' published between 2012 and 2020. We included studies in humans and animal models. Most studies reported differences in the metabolome of subjects with endometriosis in comparison to healthy controls and used samples taken from serum, endometrial tissue, follicular fluid, urine, peritoneal fluid, or endometrial fluid. Statistically significant metabolites contributed to group separation between patients and healthy controls. Reported metabolites included amino acids, lipids, organic acids, and other organic compounds. Differences in methods, analytical techniques, and the presence of confounding factors can interfere with results and interpretation of data. Metabolomics seems to be a promising tool for identifying significant metabolites in patients with endometriosis. Nonetheless, more investigation is needed in order to understand the significance of the study results.

Lay summary

Endometriosis is a chronic disease affecting the quality of life in one out of every ten women during their reproductive years, causing pain and infertility. It is characterized by inflammation and growth of tissue like the endometrium (uterus lining) outside the uterine cavity. Studies have searched for a predictor of endometriosis-associated changes by observing small molecules necessary for metabolism on a large scale (metabolomics). Metabolomics could serve to resolve one of the biggest challenges that patients with endometriosis face: a delay in diagnosis. In this review, the authors summarize identified potential biomarkers from various bodily fluids and tissues that are characteristic of metabolic processes observed in endometriosis. Biomarkers include cell growth, cell survival, high energy demand, oxidative stress, and fatty acid levels. A metabolomics approach offers promise as a non-invasive tool to identify significant metabolite changes in patients with endometriosis, potentially leading to earlier diagnoses and new opportunities for back-translational strategies.

Valéria Barradas, Mariana Pereira Antoniassi, Paula Intasqui, Marcilio Nichi, Ricardo Pimenta Bertolla, and Deborah Montagnini Spaine

Varicocele, defined by a dilation of efferent testicular veins, is the most commonly identifiable, surgically correctable lesion associated with male-factor infertility, starts at puberty and causes a progressive decline in fertile potential. The pathophysiology of infertility caused by this disease is still poorly understood, but it is suggested that the main mechanism is oxidative stress. Therefore, the aim of this study was to verify if varicocele is associated with changes in enzymatic antioxidant mechanisms and seminal plasma lipid peroxidation levels in adolescents. We recruited 90 adolescents that were divided into control (C; n= 27); varicocele and normal semen (VNS; n=46); varicocele and altered semen (VAS; n=17). Seminal and serum levels of lipid peroxidation were quantified by thiobarbituric acid reactive substances (TBARS). Seminal plasma antioxidant profile was evaluated by the activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). The VAS group had increased lipid peroxidation levels when compared to the other groups. The levels of serum lipid peroxidation and activities of the enzymes SOD and GPx did not differ between groups. CAT was undetectable by the method used. In conclusion, in adolescents with varicocele and altered semen analysis, there is an increase in seminal lipid peroxidation levels compared to adolescents with varicocele and without seminal change and adolescents without evident varicocele. However, the observed oxidative stress is not caused by a decrease in superoxide dismutase and glutathione peroxidase activities, which did not differ between adolescents with and without evident varicocele.

Sarah E Melford, Anthony H Taylor, and Justin C Konje

Objective

To determine if models of human 'receptive' and 'non-receptive endometrium' differ in their responses to nitric oxide (NO) supplementation by measuring the levels of the enzymes of the endocannabinoid system (ECS) (fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD)), which control the 'anandamide tone' essential for successful pregnancy.

Design

A study of FAAH and NAPE-PLD expression (using human endometrium) through the menstrual cycle and an in vitro using a model of 'receptive' (Ishikawa) and 'non-receptive' (HEC-1A) human endometrial cell lines treated with the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP).

Results

Immunoreactivity measured by optimised H-score for both FAAH and NAPE-PLD was reduced in secretory (receptive) endometrium compared to proliferative (non-receptive) endometrium (P = 0.0009 and <0.0001, respectively). FAAH and NAPE transcript levels were significantly higher in untreated Ishikawa cells than in HEC-1A cells (P = 0.0228 and 0.0001, respectively). Treatment of cultures with SNAP resulted in an increase in the amount of FAAH mRNA produced by Ishikawa cells and a decrease in NAPE-PLD mRNA. No effect of SNAP was observed in HEC-1A cells. Similarly, FAAH protein was significantly decreased in endometria representative of the receptive endometrium.

Conclusion

These data suggest that NO most likely affects the expression of ECS enzymes in the implantation site of a receptive endometrium; a phenomenon not seen in a non-receptive endometrium. These effects are most marked with FAAH expression, suggesting that FAAH may play the more critical role in ensuring the correct 'anandamide tone' for successful embryo implantation than NAPE-PLD.

Lay summary

Embryo implantation into the wall of the uterus is only successful when the inner wall of the uterus (the endometrium) is ‘receptive’, because if it is ‘non-receptive’, implantation will fail. Previous work showed that enzymes of the 'endocannabinoid system' are critical for implantation by maintaining the correct level of a fat called anandamide. This is by balancing its synthesis (by N-acylphosphatidylethanolamine specific phospholipase D, NAPE-PLD) and degradation (by fatty acid amide hydrolase, FAAH). Using immortalised cell lines as models of ‘receptive’ and ‘non-receptive’ human endometrium, we demonstrate a key stimulator of implantation, nitric oxide, has a positive effect on implantation by both increasing the mRNA levels of the degrading enzyme (FAAH) and decreasing the expression of the synthesising enzyme (NAPE-PLD). These effects are most marked with the degrading enzyme, suggesting that FAAH plays a more critical role than NAPE-PLD in ensuring the correct 'anandamide tone' for successful embryo implantation.

Roberto Rodrigues da Rosa Filho, Maíra Morales Brito, Thaís Gomes Faustino, Leticia Lima de Almeida, Verônica Correia Manoel, Bruno Cogliati, and Camila Infantosi Vannucchi

Effects of conservative treatment on uterine blood flow and morphometric findings are still unknown in bitches. Thus, this study aimed to compare uterine changes of pyometra bitches subjected to distinct modes of treatment. Pyometra bitches were assigned to: OHE (ovariohysterectomy immediately after diagnosis), Aglepristone (days 1, 2 and 8) and Associative (aglepristone treatment coupled with cloprostenol for 7 days) groups. After 9 days, bitches were ovariohysterectomized. Before surgery, uterine area was measured ultrasonographically and the uterine artery Doppler velocimetry analyzed blood flow velocity and indexes. Uterine horns were classified according to resistance index (RI) as more compromised and less compromised. Endometrial vasculature was quantitatively evaluated by color flow Doppler. Blood samples were collected to determine nitric oxide (NO) concentrations. Histological uterine structures were quantified by stereology and VEGF-A (vascular endothelial growth factor) and eNOS were (endothelial nitric oxide synthase) immunohistochemically analyzed. Aglepristone and Associative groups had lower uterine area and vascularization, and higher blood flow velocity and indexes compared to OHE group. Less compromised horn of Associative group had higher blood flow velocity compared to OHE group. Aglepristone group presented lower inflammatory infiltrate and larger uterine stroma. Associative group had lower volume density and absolute surface of endometrial cysts and lower VEGF-A expression for glandular epithelium and stromal cells. Blood NO and e-NOS immunostaining were not different among groups. In conclusion, association between aglepristone and prostaglandin is more effective in decreasing uterine vascularization and modulating uterine blood flow. Moreover, associative therapy promotes marked morphological changes.

Lay summary

This research compared two medical protocols of treatment for uterine infection (pyometra) in bitches, using a hormone blocker (anti-progesterone aglepristone) solely or in association with a uterine contraction inducer (prostaglandin; associative therapy). After treatment, bitches were gonadectomized and a microscopic analysis of uterine blood vessel formation and uterine tissue elements were performed as well as uterine blood flow evaluation through Doppler ultrasonography. According to vascular resistance, uterine horns were additionally classified as more compromised and less compromised. Both treatment protocols led to reduction of uterine dimensions and vascularization, and higher blood flow compared to untreated bitches. Less compromised uterine horn of the associative treatment had higher blood flow compared to untreated bitches. The hormone blocker treatment had lower inflammatory cells and larger uterine histological structure, while associative treatment had less uterine pathological cysts and lower blood vessel formation. The associative therapy is effective in decreasing uterine vascularization and modulating uterine blood flow as well as reestablishing endometrium structure in bitches with uterine infection.

Iman Al-Saleh, Serdar Coskun, Reem Al-Rouqi, Tahreer Al-Rajudi, Chafica Eltabache, Mai Abduljabbar, and Saad Al-Hassan

This study examined the status of oxidative stress in 599 couples undertaking in vitro fertilization (IVF) treatment and its association with reproductive hormones, smoking, and outcomes. Oxidative stress biomarkers such as malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), hydrogen peroxide (H2O2), catalase (CAT), and total antioxidant capacity (TAC) were determined in follicular fluid and seminal plasma. Tail moment (TM) was used to evaluate DNA damage in sperm and granulosa cells. Reproductive hormones in serum and cotinine (COT) in urine, follicular fluid, and seminal plasma samples were determined. We used log-binomial multivariate regression to estimate relative risks for the association between oxidative stress/DNA damage and IVF binary outcomes (fertilization rate, biochemical pregnancy, clinical pregnancy, and live birth). We observed an increase in the oxidative stress markers MDA, 8-OHdG, and H2O2 in follicular fluid and seminal plasma, but a decrease in the antioxidant protection markers CAT and TAC. The MDA, 8-OHdG, and H2O2 levels were significantly higher in seminal plasma than in follicular fluid, while TAC, CAT, and TM were higher in follicular fluid (p < 0.001). Although women were nonsmokers, COT levels >50 µg/l were observed in 5.7% (urine) and 1.4% (follicular fluid). An increase in the CAT levels of follicular fluid was associated with a 48 and 41% decrease in the risk of poor fertilization rate (≤50%) and unsuccessful live birth, respectively. After the models were adjusted for hormonal factors, the associations remained the same, except that elevated TAC in follicular fluid became significantly associated with a decrease of 42% in the risk of poor fertilization rate (≤50%).

Briet D Bjarkadottir, Charlotte A Walker, Muhammad Fatum, Sheila Lane, and Suzannah A Williams

In vitro follicle growth is a potential fertility preservation method for patients for whom current methods are contraindicated. Currently, this method has only been successful using fresh ovarian tissue. Since many patients who may benefit from this treatment currently have cryopreserved ovarian tissue in storage, optimising in vitro follicle growth (IVG) for cryopreserved-thawed tissue is critical. This study sought to improve the first step of IVG by comparing different short-term culture systems for cryopreserved-thawed human ovarian tissue, in order to yield a higher number of healthy multilayer follicles. We compared two commonly used culture media (αMEM and McCoy’s 5A), and three plate conditions (300 µL, 1 mL on a polycarbonate membrane and 1 mL in a gas-permeable plate) on the health and development of follicles after 6 days of culture. A total of 5797 follicles from three post-pubertal patients (aged 21.3 ± 2.3 years) were analysed across six different culture conditions and non-cultured control. All culture systems supported follicle development and there was no difference in developmental progression between the different conditions tested. Differences in follicle morphology were evident with follicles cultured in low volume conditions having significantly greater odds of being graded as morphologically normal compared to other conditions. Furthermore, culture in a low volume of αMEM resulted in the highest proportion of morphologically normal primary and multilayer follicles (23.8% compared to 6.3-19.9% depending on condition). We, therefore, recommend culturing cryopreserved human ovarian tissue in a low volume of αMEM to support follicle health and development.

Lay summary

Ovaries contain a large number of follicles, each containing an immature egg and other important cells. Cancer treatments can lead to long-lasting negative side effects to the ovaries including the destruction of follicles, resulting in infertility. One strategy to preserve fertility is freezing of ovaries or ovarian tissue in girls and women undergoing cancer treatment. The long-term aim is to thaw and grow their ovarian tissue in the laboratory to obtain mature eggs, which can then be fertilised. In this study, we compared six different methods of growing previously frozen human ovarian tissue in order to best support follicle growth and health. We found that using the lowest amount of αMEM medium (a specific type of nutrient-rich growth solution) resulted in the highest proportion of healthy follicles. Improving the methods used to grow ovarian tissue, particularly frozen tissue, is important for future fertility preservation.

Jennifer B Nagashima, Andrea M Hill, and Nucharin Songsasen

Graphical Abstract

Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts.

Lay summary

The ovary contains hundreds of eggs with only a select few developing from an immature stage through to ovulation over the course of an animal's lifetime. Rescue of eggs from this pool, and the ability to grow them in culture to a mature stage, would be incredibly valuable for fertility preservation efforts in both humans and endangered species. Currently, the isolation of ovarian follicles (eggs with their surrounding helper cells) is a key step in culture systems for large mammalian species, to promote continued growth. Yet, isolation methods may affect the follicle’s future developmental capacity. We evaluated two isolation strategies, mechanical micro-dissection (needle/scalpel blade) and enzymatic digestion (using Liberase blendzyme) on ovaries of domestic cats obtained via routine spay procedures. Mechanically isolated follicles displayed improved growth, survival, and indications of developmental competence in 14-day culture, compared with high concentration (1.4 Wünsch units/mL) enzyme-isolated follicles. However, mechanical isolation was not different from low (0.7 Wünsch units/mL) enzyme for these metrics, or for expression of key genes indicative of follicular cell functions. Further, differences in follicle growth/survival were not apparent until 7+ days in culture. Thus, ovarian follicle isolation strategies influence developmental potential in culture, and extended culture will be required to identify optimal methods for fertility preservation efforts.

Douglas A Gibson, Frances Collins, Bianca De Leo, Andrew W Horne, and Philippa T K Saunders

Endometriosis is a chronic neuroinflammatory pain condition affecting ~180 million women worldwide. Surgical removal or hormonal suppression of endometriosis lesions only relieves pain symptoms in some women and symptomatic relapse following treatment is common. Identifying factors that contribute to pain is key to developing new therapies. We collected peritoneal fluid samples and clinical data from a cohort of women receiving diagnostic laparoscopy for suspected endometriosis (n = 52). Peritoneal fluid immune cells were analysed by flow cytometry and data compared with pain scores determined using the pain domain of the Endometriosis Health Profile Questionnaire (EHP-30) in order to investigate the association between peritoneal immune cells and pain symptoms. Pain scores were not different between women with or without endometriosis, nor did they differ according to disease stage; consistent with a poor association between disease presentation and pain symptoms. However, linear regression and correlation analysis demonstrated that peritoneal macrophage abundance correlated with the severity of pelvic pain. CD14high peritoneal macrophages negatively correlated with pain scores whereas CD14low peritoneal macrophages were positively correlated, independent of diagnostic outcome at laparoscopy. Stratification by pain subtype, rather than endometriosis diagnosis, resulted in the most robust correlation between pain and macrophage adundance. Pain score strongly correlated with CD14high (P = 0.007) and CD14low (P = 0.008) macrophages in patients with non-menstrual pain and also in patients who reported dysmennorhea (CD14high P = 0.021, CD14low P = 0.019) or dysparunia (CD14high P = 0.027, CD14low P = 0.031). These results provide new insight into the association between peritoneal macrophages and pelvic pain which may aid the identification of future therapeutic targets.

Lay summary

Endometriosis is a common condition where cells similar to those that line the womb are found elsewhere in the body. It is associated with inflammation and pain in the pelvis and affects ~180 million women worldwide. Current treatments are not effective for all patients and we, therefore, need to understand what causes pain in order to develop new treatments. We investigated the types of immune cells present within the pelvis of women undergoing investigation for suspected endometriosis. Disease diagnosis and stage (I–IV) was recorded along with pain score determined by questionnaire. We characterised the immune cells present and compared them to disease stage and pain score. We found that pelvic pain was linked to the abundance of immune cells but, surprisingly, not to disease stage. These findings suggest that immune cells are closely associated with pain severity in endometriosis and may be good targets for future endometriosis treatments.