Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs.

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus-oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analysed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P = 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase.

Effect of two 'progressively motile sperm-oocyte' ratios on porcine in vitro fertilization and embryo development.

Sperm motility and viability of cryopreserved semen vary between boars and straws, which influences the outcomes of in vitro embryo production (IVEP). However, progressive motility is usually not considered during IVEP. The aim of this study was to assess fertilization with a 500:1 and 250:1 'progressively motile sperm to oocyte' ratio on IVEP outcomes using semen from three Duroc and three Landrace boars. Frozen-thawed sperm was centrifuged through a 45/90% Percoll® density gradient and sperm quality parameters were assessed. In vitro matured oocytes were fertilized at the two ratios, a portion was stained 10-12 h after start of fertilization to analyze fertilization and polyspermy, while the remaining zygotes were cultured up to day 7. The 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared with the 250:1 ratio, but no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates, but not for the other IVEP outcomes. In conclusion, a higher fertilization and blastocyst yield was obtained with the 500:1 ratio compared with the 250:1 ratio, while polyspermy level was consistent across ratios. Differences in IVEP outcomes were still observed between the individual boars although adjusted for progressive motility. Promising blastocyst yields and high total blastocyst cell counts were obtained with sperm from both breeds.

Effect of Previous Alkylating Agent Exposure on Follicle Numbers in Cryopreserved Prepubertal and Young Adult Ovarian Tissue after Long-Term Xenografting.

PURPOSE AND METHODS: To elucidate whether previous cancer treatment affects graft recovery and follicle numbers, morphology, and development in grafts, cryopreserved ovarian biopsies obtained from 18 cancer patients aged 1-24 years with and without exposure to chemotherapy were xenografted as 1 mm3 fragments to immunodeficient mice for 22 weeks with exogenous stimulation. RESULTS: Graft recovery showed no association with chemotherapy exposure, pubertal stage, or leukemia contamination. Total follicle number per recovered graft varied between 0 and 1031 in the chemotherapy-exposed and between 0 and 502 in the non-chemotherapy-exposed group. Atretic follicles formed the largest proportion of the follicle pool in chemotherapy-exposed grafts. Increased atresia correlated with exposure to alkylating agents (mean ± SD 8866.2 ± 9316.3 mg/m2) but not with anthracyclines, pubertal stage, or leukemia contamination. CONCLUSION: The observation confirms the harmful effects of alkylating agents on ovarian tissue. Therapy at the median cumulative dose of 8866 mg/m2 leads to the decreased quality of cryopreserved ovarian follicles in children and young adults.

Ovarian characteristics and in vitro nuclear and cytoplasmic oocyte maturation in Duroc and Landrace pigs.

Differences in total number of piglets born per litter are observed between the Norwegian Duroc (ND) sire and Norwegian Landrace (NL) dam line. The aim of this study was to evaluate ovarian characteristics, and in vitro nuclear and cytoplasmic oocyte maturation in both breeds. One day after weaning, follicular phase ovaries were collected. Ovary length and weight were measured and the number of follicles (< 3 mm and 3-8 mm) was counted. Cumulus-oocyte complexes (COCs) were collected and matured for 48 hr. To assess cumulus expansion, COC area was analysed at 0 and 20 hr. Nuclear maturation and cortical granule (CG) distribution were analysed at 20 and 48 hr, and total glutathione (GSH) was measured at 48 hr to further elucidate cytoplasmic maturation. In first parity sows, a smaller ovary length and fewer 3 to 8 mm follicles were observed in ND compared to NL. For all sows, ND COCs covered a significantly smaller area at 0 hr, but a higher cumulus expansion ratio was observed at 20 hr compared to NL (364 ± 46% versus. 278 ± 27%, p < 0.001). At 20 hr, more ND oocytes exhibited advanced stages of nuclear maturation, while more NL oocytes showed advanced stages of CG distribution. Nuclear maturation to MII stage at 48 hr did not differ between ND and NL oocytes (90.1% and 87.7%, respectively). Moreover, no significant differences were observed for GSH content or CG distribution after maturation. In conclusion, differences with regard to ovarian characteristics as well as to cumulus expansion, and nuclear and cytoplasmic oocyte maturation at 20 hr were observed between the breeds. Further studies are required to determine if this subsequently affects in vitro fertilization and embryo development.

Micromorphological and ultrastructural description of spermatozoa from squirrel monkeys (Saimiri collinsi Osgood, 1916).

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

Impact of first-line cancer treatment on the follicle quality in cryopreserved ovarian samples from girls and young women.

STUDY QUESTION: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients? SUMMARY ANSWER: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment. WHAT IS KNOWN ALREADY: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients. WIDER IMPLICATION OF THE FINDINGS: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare.

Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

Minimal residual disease of leukemia and the quality of cryopreserved human ovarian tissue in vitro.

Auto-transplant of cryopreserved ovarian tissue in leukemia patients carries a risk to reintroduce malignant cells. Maturation of ovarian follicles in vitro is a promising strategy to overcome the leukemic cell contamination. The follicle development and survival in 14 cryopreserved ovarian tissues with leukemia-specific PCR marker was evaluated after 7 or 14 days culture. Minimal residual disease (MRD) quantification was assessed by real-time quantitative PCR in order to identify the MRD positive (n = 6) and negative (n = 8) samples and to monitor levels of MRD before and after culture. The morphology of ovarian follicles were studied by light microscopy. After culture, no statistical significant differences were detected in follicle densities between MRD positive- and negative samples. Ovarian MRD either decreased below undetectable or fluctuated near the baseline level after 7 and 14 days in culture. This study provides quantitative in vitro evidence that leukemia contamination does not affect the follicle survival in cryopreserved ovarian tissue.

Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro.

BACKGROUND: Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance. MATERIALS: In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35), cryopreserved for fertility preservation before (n = 14) or after (n = 20) initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED) were tested. RESULTS: Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles. CONCLUSION: This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer developing follicles in culture.

Trolox enhances follicular survival after ovarian tissue autograft in squirrel monkey (Saimiri collinsi).

The aim of this study was to evaluate ovarian tissue pre-treatment with 50 µM Trolox followed by heterotopic transplantation in squirrel monkeys (Saimiri collinsi) and to assess tissue functionality via immunohistochemical analysis of the stroma and ovarian follicles. Five healthy and sexually mature squirrel monkey (Saimiri collinsi) females were used. Heterotopic autografting of fresh ovarian tissue with or without previous exposure to the antioxidant Trolox was performed and grafts were recovered for analysis 7 days later. Tissue vascularisation was confirmed by both macroscopic inspection and cluster of differentiation 31 (CD31) staining. Trolox prevented massive follicular activation and kept the percentages of morphologically normal follicles higher than in untreated grafts. Expression of anti-Müllerian hormone in developing follicles was observed only in controls and Trolox-treated grafts. Also, immunostaining for growth differentiation factor-9 was positive only in primordial follicles from controls and from Trolox-treated grafts. Although Trolox improved follicular quality and avoided apoptosis in stromal cells, ovarian tissue fibrosis was increased in Trolox-treated grafts, mainly due to an increase in collagen Type I synthesis.

Vitrification of ovarian tissue from primates and domestic ruminants: an overview.

In the last decade, vitrification protocols to preserve human ovarian tissue have been regularly reported, even more often than the protocols developed for large mammals, such as ruminants and nonhuman primates. In order to facilitate the use of domestic ruminants (cows, goats, and sheep) and nonhuman primates as animal models, application of similar protocols as used for human material is performed. Next to it, the addition of indispensable or exclusion of avoidable compounds in the vitrification of human ovarian tissue should be tested in such experiments with animal models. The objective of this mini-review is to summarize the current protocols used for the vitrification of ovarian tissue and to evaluate the vitrification methods in humans, nonhuman primates, and domestic ruminants.

Fertility-preserving measures for girls and young women with cancer.

BACKGROUND: Children and young adults with cancer may be rendered infertile as a result of their treatment. The purpose of this article is to provide an overview of fertility-preserving measures for girls and young women. MATERIAL AND METHODS: The article is based on literature searches in the medical databases Medline, Pubmed and Scopus and the experience of a Nordic cooperative group on gonadal preservation in connection with cancer treatment. RESULTS: There are several methods for preserving the fertility of girls and young women with cancer. These should form a part of the actual cancer treatment. Cryopreservation of embryos is a well established method for adult cancer patients, also in Norway. Cryopreservation of eggs and ovarian tissue is to be regarded as still at the experimental stage. Research and new methods will improve the options for prepubertal children and young adults with disseminated cancer. INTERPRETATION: Multidisciplinary cooperation is necessary to ensure that children and young cancer patients receive thorough information about the risk of infertility after cancer treatment, and about potential fertility-preserving measures.

Fertility-preserving measures for boys and young men with cancer.

BACKGROUND: Some types of cancer treatment entail a risk of reduced fertility and infertility. Fertility-preserving treatment can reduce the risk for some. The purpose of this article is to provide an overview of the risk of infertility after treatment of boys and young men with cancer and of fertility-preserving measures. MATERIAL AND METHODS: The article is based on literature searches in the medical databases Medline, Pubmed and Scopus and on the experience of a Nordic medical network collaboration. RESULTS: Cryopreservation of sperm is an established method for adult cancer patients in Norway. Vibratory stimulation of the penis and electroejaculation with subsequent freezing of sperm may be an option for young cancer patients who cannot manage to produce a semen sample with the aid of masturbation. Freezing of testicular biopsies may be an option for prepubertal boys who are not capable of producing mature sperm. INTERPRETATION: There are established methods for cryopreservation of sperm for adult cancer patients. The other fertility-preserving measures for boys and young men with cancer are regarded as experimental at present.

Histologic and ultrastructural features of cryopreserved ovine ovarian tissue: deleterious effect of 1,2-propanediol applying different thawing protocols.

Ovarian cortical fragments were frozen in presence of 1.5 M PROH, thawed at different temperatures (37 degrees, 30 degrees, or 4 degrees C) and submitted to histologic and ultrastructural analysis. Thawing at 30 degrees C minimized stromal and follicular damage when compared with 4 degrees and 37 degrees C.

Altered stress-induced cortisol levels in goats exposed to polychlorinated biphenyls (PCB 126 and PCB 153) during fetal and postnatal development.

Short-term stress exposure is associated with activation of the hypothalamic-pituitary-adrenal (HPA) axis and a consequent rise in blood glucocorticoids and catecholamines, from the adrenal cortex and medulla, respectively. The HPA axis is a potential target for some persistent organic pollutants, among which polychlorinated biphenyls (PCB) were found to be modulators of the mammalian endocrine system. PCB are distributed globally in the environment, in food chains, and are transferred to the fetuses of pregnant animals and via mother's milk to suckling offspring. In the present study it was postulated that intrauterine and lactational exposure to either of two single congeners of PCB (PCB 153 and PCB 126, respectively) might affect basal cortisol concentrations, and also the cortisol response to short-term stress in adulthood. Thus, pregnant goats were orally exposed to one of these PCB congeners from d 60 of gestation until delivery, and their offspring studied. Low-dose exposure to PCB 153 and PCB 126 resulted in significantly lower mean basal cortisol concentrations in goat offspring during certain periods of pubertal development and their first breeding season. Male goat kids exposed to either PCB congener showed a greater and more prolonged rise in plasma cortisol levels than controls when animals were subjected to mild stress at 9 mo of age using frequent blood sampling. Neither the basal maternal cortisol plasma level nor goat kid adrenal masses were affected by PCB exposure.

Endocrine disruption induced by organochlorines (OCs): field studies and experimental models.

Long-range transport of persistent organic compounds by air and ocean currents from industrialized areas resulted in high levels of these pollutants in food webs in the Svalbard area. With the aim to test if organochlorine (OC) exposure in free-living polar bears from Svalbard affected their plasma steroid hormone concentrations, it was found that polychlorinated biphenyls (PCBs) were associated with increased progesterone levels in females. The sum of pesticides (sigma pesticides) and sigma PCBs contributed significantly negative to the variation of the plasma testosterone in males, and the overall contribution of the OCs to the plasma cortisol variation was negative. A second objective was to study the effects of selected OCs (i.e., PCB 153 and PCB 126) on animal health as a consequence of effects on endocrine-regulated functions such as reproduction and immunity in a goat model focusing on long-term and low-level exposure during the periods of fetal development and in the neonatal period. Additionally, acute exposure was studied in adult mice. The results indicated that exposure to low doses of PCB 153 in utero and in the suckling period influenced reproductive functions and both PCB 153 and PCB 126 exerted immunomodulatory effects on the offspring, whereas acute exposure of adult mice had minor effects on male reproductive function.

Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats.

In this study, female goats were orally exposed to PCB126 or PCB153, at 49 ng/kg body weight per day and 98 microg/kg body weight per day respectively, from gestational day 60 until delivery at approximately day 150. Exposure of the offspring continued via lactation until postnatal day 40. Reproductive toxicity in the male offspring was studied by the evaluation of conventional reproductive endpoints as well as flow cytometric analyses of spermatogenesis and sperm chromatin structure. PCB153-treated animals showed a significant smaller testis diameter in comparison to the control group. Neither of the treated groups showed differences for plasma FSH in comparison to controls. PCB153-treated animals differed significantly from the control group with respect to plasma LH and testosterone levels, whereas PCB126-treated animals only differed from the controls in plasma testosterone concentrations. Neither the PCB126 nor the PCB153 group differed from the controls with respect to the conventional sperm parameters or testis histology. A significant lower ratio of interstitium area to seminiferous tubules area and proportion of diploid testis cells were observed for the PCB153 group. Sperm from PCB153-treated animals showed a significantly higher percentage of sperm with damaged DNA. From the results of the present study it was concluded that PCB153 was able to induce alterations in reproductive endpoints related to the hypothalamic-pituitary-axis as well as to the testis. The effects observed in male kids after a long-term maternal exposure to PCB153 support the concept that exposure to endocrine-disrupting compounds during foetal development may lead to adverse reproductive effects in adult life.

Effects of PCB99 and PCB153 exposure on spermatogenesis in young adult C57BL6 mice.

This study examined the effects of acute exposure to PCB99 (2,2',4,4',5-pentachlorobiphenyl), and PCB153 (2,2',4,4'5,5'-hexachlorobiphenyl), on spermatogenesis in 8-week-old C57BL6 mice. The mice were randomly allocated to PCB99 and PCB153 and a single dose of respectively 10 and 100 mg/kg was given by oral gavage. During the 6-week experiment, six mice per treatment group were sacrificed weekly, body weights were recorded and samples with respect to the male reproductive system were collected until further analysis. None of the treatments, showed changes in body weight or reproductive endpoints. Flow cytometric analysis revealed spermatogenesis to be unaffected. However, PCB99 and PCB153 showed a significant increase in Leydig cell apoptosis. The results from the present study indicate that the male reproductive system is relatively refractory to PCB99 and PCB153 at levels exceeding those of wildlife and humans, when exposed during adult life. However, the finding of apoptotic Leydig cells merits further investigation.

Effects of gestational and lactational exposure to low doses of PCBs 126 and 153 on anterior pituitary and gonadal hormones and on puberty in female goats.

The aim of the present study was to investigate if environmental doses of PCB 153 and PCB 126 could produce effects in a controlled animal model. Possible adverse effects on the hypothalamic-pitutitary-gonadal axis were examined by measuring gonadotrophins and gonadal steroid hormone concentrations in goat kids exposed during gestation and lactation. The concentrations of PCB 153 and PCB 126 in adipose tissue in the goat kids 9 months post-partum were 5800 ng/g (fat-weight, range; 2900-12700 ng/g) and 0.49 ng/g (fat-weight, range; 0.28-0.80 ng/g), respectively. The pre- and post-pubertal plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (Prl) and progesterone (P4) were analysed. LH, FSH, Prl, and P4 were also measured during an induced oestrus cycle. The prepubertal LH concentration was significantly lower, the puberty was delayed and the P4 level during the luteal phase of an estrous cycle was higher in the group exposed to PCB 153. No significant effect of PCB 153 exposure was found on Prl and FSH. PCB 126 did not produce any effects at the exposure level tested in this study. In conclusion, perinatal exposure to PCB 153 affected the reproductive function and the puberty maturation in goats.