Human Oocyte Morphology and Outcomes of Infertility Treatment: a Systematic Review.

Oocyte morphology assessment is easy to implement in any laboratory with possible quality grading prior to fertilization. At present, comprehensive oocyte morphology scoring is not performed as a routine procedure. However, it may augment chances for successful treatment outcomes if a correlation with certain dysmorphisms can be proven. In order to determine a correlation between oocyte morphology and treatment outcome, we performed a systematic search in PubMed and Cochrane Controlled Trials Register following PRISMA guidelines. A total of 52 articles out of 6,755 search results met the inclusion criteria. Dark colour of the cytoplasm (observed with an incidence rate of 7%), homogeneous granularity of the cytoplasm (19%) and ovoid shape of oocytes (7%) appeared to have no influence on treatment outcome. Abnormalities such as refractile bodies (10%), fragmented first polar body (37%), dark zona pellucida (9%), enlarged perivitelline space (18%) and debris in it (21%) are likely to affect the treatment outcome to some extent. Finally, cytoplasmic vacuoles (4%), centrally located cytoplasmic granularity (12%) and clusters of smooth endoplasmic reticulum (4%) negatively impact infertility treatment outcomes. Nonetheless, morphological assessment is informative rather than predictive. Adding oocyte morphology to the artificial intelligence (AI)-driven selection process may improve the precision of the algorithms. Oocyte morphology assessment can be especially useful in oocyte donation cycles, during oocyte freezing for fertility preservation and finally, objective oocyte scoring can be important in cases of very poor treatment outcome as a tool for explanation of results to the patient.

Physical and Sedentary Activities in Association with Reproductive Outcomes among Couples Seeking Infertility Treatment: A Prospective Cohort Study.

BACKGROUND: The aim of this study was to investigate the association of physical activity (PA) with assisted reproductive technology (ART) treatment and pregnancy outcomes among couples seeking infertility treatment. METHODS: This prospective cohort study was carried out among 128 infertile individuals (64 couples), entering the infertility clinic for ART procedures. Baseline PA (before entering any treatment) was assessed using accelerometry for both women and men. For every couple the infertility treatment outcomes were recorded. RESULTS: The couples that required invasive ART procedures such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) spent less time in vigorous PA (-73 min/week per couple, woman + man) than those couples who became spontaneously pregnant after entering the study (p = 0.001). We observed no significant associations between the time spent in physical activities and positive pregnancy test or live birth. CONCLUSIONS: Our results do not support a positive nor negative relation between the time the couples spent in physical activities and the chances of getting pregnant or having a baby among patients seeking infertility treatment. However, couples undergoing invasive ART procedures did less vigorous PA than couples that became spontaneously pregnant, suggesting that PA may interfere with their reproductive health.

Assisted hatching of vitrified-warmed blastocysts prior to embryo transfer does not improve pregnancy outcomes.

OBJECTIVE: This study aims to determine the impact of assisted hatching (AH) on pregnancy outcomes in vitrified-warmed blastocyst transfers, and evaluate if embryo expansion or morphology influences outcomes. METHODS: A retrospective cohort study was performed including vitrified-warmed blastocyst transfers at our clinic between 2013 and 2017. Of the 2165 embryo transfers, 1986 underwent laser AH and 179 were non-assisted hatched (NAH). The primary outcome was live birth. Secondary outcomes included conception, implantation, clinical pregnancy, clinical pregnancy loss, and monozygotic twinning (MZT). RESULTS: AH and NAH groups had similar rates of conception (38.7% vs 42.1%), implantation (26.2% vs 27.3%), clinical pregnancy (29.1% vs 30.3%), clinical pregnancy loss (24.0% vs 17.8%), live birth (19.9% vs 20.5%), and MZT (2.08% vs 2.86%). Five pairs of dichorionic/diamniotic twins resulted from single embryo transfers. AH of embryos with expansion grades ≤3 was associated with lower rates of conception (32.5% vs 44.3%%, p < 0.05) and clinical pregnancy (24.0% vs 32.8%, p < 0.05). CONCLUSION: AH prior to transfer of vitrified-warmed blastocysts was not associated with improved pregnancy outcomes. The identification of dichorionic/diamniotic twins from single blastocyst transfers challenges the previously held notion that dichorionic/diamniotic MZTs can only occur from division prior to the blastocyst stage. Prospective studies are needed to validate the novel finding of lower rates of conception and clinical pregnancy after AH in embryos with lower expansion grade.

Preimplantation genetic testing practices in the Nordic countries.

INTRODUCTION: Preimplantation genetic testing (PGT) is growing in importance and volume internationally. International societies such as the European Society for Human Reproduction and Embryology compile international results and these data are published in scientific journals. We present the first compilation of practices, quality measuress and outcome data from Nordic clinics performing PGT. MATERIAL AND METHODS: We conducted a structured online survey of PGT practices in the Nordic countries to compare clinical and laboratory techniques, outcomes and quality measures applied in Nordic clinics. The survey was designed by the authors and answered by the authors and members of the study group. The outcome data represents results from 2018. Results and details were clarified through iteration with responding clinics while maintaining anonymity. Response rate in the study was 80%, with 8 of 10 clinics performing PGT responding. RESULTS: Most of the PGT cycles in the Nordic countries are funded through the public healthcare system with University Hospitals performing the majority of treatments, 716/848, or 84.4%, of oocyte retrievals in this dataset. The genetic analyses are in five cases performed by the affiliated local genetic laboratory, and the remaining three consult with large international private enterprise laboratories. Genetic counseling is widely used. Results in the Nordic clinics compare well with international data. Systematic quality control procedures are in place and the larger clinics and laboratories utilize ISO certification or accreditation in the quality management. Automatic witnessing with detailed electronic documentation of laboratory processes is not utilized in the responding clinics, although a majority uses manual witnessing procedures in the laboratory. The outcome after PGT in terms of clinical pregnancy per transfer is around 40% per embryo transfer and compares well with international data. CONCLUSIONS: Preimplantation genetic testing is organized in rather few clinics in the Nordic countries and most of them use local laboratories for genetic analyses of the biopsies. Laboratory procedures are largely in accordance with international guidelines and the outcome after PGT in terms of clinical pregnancy per transfer is comparable to results in international reports.

Preimplantation genetic testing legislation and accessibility in the Nordic countries.

INTRODUCTION: Assisted reproduction technologies are being rapidly developed and implementation of preimplantation genetic testing (PGT) has allowed patients with genetic disorders to initiate pregnancies while minimizing or eliminating the risk of transmitting these disorders to their offspring. Testing for numeric chromosomal anomalies has been proposed as a way to increase efficacy in assisted reproduction; however, this remains disputed. Legislation is lagging behind the rapid developments in this field. MATERIAL AND METHODS: We conducted a structured online survey of legislation and accessibility to preimplantation genetic testing in the Nordic countries to compare the regulation and uptake of this technique. The survey was designed and answered by the authors. RESULTS: Key elements in the regulation of preimplantation testing for monogenic disorders and structural rearrangements are similar in the Nordic countries, although accessibility varies since only Denmark, Finland, and Sweden have national clinics offering treatment. In addition, Denmark and Finland have private clinics offering PGT. Regulation is the most stringent in Norway where a national board evaluates all couples seeking treatment. Treatment volumes vary between the Nordic countries, with Norway and Finland having lowest treatment numbers. Preimplantation genetic testing for aneuploidy in the embryo varies between the Nordic countries: Finland and Iceland allow this form of treatment, Denmark and Sweden offer it only in the form of a research protocol, and Norway does not allow it at all. Therefore the number of treatment cycles involving testing for embryo aneuploidy are lower in the Nordic countries than in other countries where this treatment option is more common. CONCLUSIONS: Science needs to inform politics regarding the rapidly evolving field of reproductive medicine and we recommend harmonization of legislation and accessibility between the Nordic countries.

Levels of caspase-3 and histidine-rich glycoprotein in the embryo secretome as biomarkers of good-quality day-2 embryos and high-quality blastocysts.

Morphological assessment at defined developmental stages is the most important method to select viable embryos for transfer and cryopreservation. Timing of different developmental stages in embryo development has been shown to correlate with its potential to develop into a blastocyst. However, improvements in pregnancy rates by using time-lapse techniques have been difficult to validate scientifically. Therefore, there is a need for new methods, preferably non-invasive methods based on metabolomics, genomics and proteomics, to improve the evaluation of embryo quality even further. The aim of this study was to investigate if different levels of caspase-3 and histidine-rich glycoprotein (HRG), secreted by the embryo into the culture media, can be used as biomarkers of embryo quality. In this study, a total of 334 samples of culture media were collected from in vitro fertilization (IVF) treatments at three different clinics. Protein analysis of the culture media was performed using multiplex proximity extension protein analysis to detect levels of caspase-3 and HRG in the embryo secretome. Protein levels were compared in secretome samples from high- and low-quality blastocysts and embryos that became arrested during development. Correlation between protein levels and time to morula formation was also analyzed. Furthermore, protein levels in secretomes from day-2 cultured embryos were compared on the basis of whether or not pregnancy was achieved. The results showed that caspase-3 levels were lower in secretomes from high-quality vs. low-quality blastocysts and those that became arrested (p ≤ 0.05 for both). In addition, higher HRG levels correlated with a shorter time to morula formation (p ≤ 0.001). Caspase-3 levels were also lower in secretomes from day-2 cultured embryos resulting in a pregnancy vs. those that did not (p ≤ 0.05). Furthermore, it was shown that caspase-3 might be used as a marker for predicting potential success rate after transfer of day-2 cultured embryos, where a caspase-3 cutoff level of 0.02 gave a prediction probability of 68% (p = 0.038). In conclusion, in future prediction models, levels of caspase-3 and HRG might be used as potential markers of embryo quality, and secreted caspase-3 levels could to some extent predict the outcome after transfer of day-2 cultured embryos.

Differences in secretome in culture media when comparing blastocysts and arrested embryos using multiplex proximity assay.

OBJECTIVES: The aim of this study was to assess different patterns of the human embryo secretome analysed as protein levels in culture media. Furthermore, analyses to correlate protein levels with quality and timing to development of human embryos were performed. MATERIAL AND METHODS: Human day-2 cryopreserved embryos were cultured for four days in an EmbryoScope® with a time-lapse camera, and embryo quality was evaluated retrospectively. After culture, the media were collected and relative levels of secreted proteins were analysed using Proseek Multiplex Assays. Protein levels were evaluated in relation to timing to development and the ability to form a blastocyst. RESULTS: Specific patterns of timing of development of blastocysts were found, where a difference in time to start of cavitation was found between high- and low-quality blastocysts. There appeared to be a correlation between specific protein patterns and successful formation of morulae and blastocysts. Embryos developing into blastocysts had higher levels of EMMPRIN than arrested embryos, and levels of caspase-3 were lower in high- versus low-quality blastocysts. Also, higher levels of VEGF-A, IL-6, and EMMPRIN correlated with shorter times to morula formation. CONCLUSIONS: The secretome and timing to development differ in embryos forming blastocysts and those that become arrested, and in high- versus low-quality blastocysts. The levels of certain proteins also correlate to specific times to development.

A prospective investigation of perceived stress, infertility-related stress, and cortisol levels in women undergoing in vitro fertilization: influence on embryo quality and clinical pregnancy rate.

INTRODUCTION: Women undergoing fertility treatment experience high levels of stress. However, it remains uncertain if and how stress influences in vitro fertilization (IVF) cycle outcome. This study aimed to investigate whether self-reported perceived and infertility-related stress and cortisol levels were associated with IVF cycle outcomes. MATERIAL AND METHODS: A prospective cohort of 485 women receiving fertility treatment was recruited from September 2011 to December 2013 and followed until December 2014. Data were collected by online questionnaire prior to IVF start and from clinical charts. Salivary cortisol levels were measured. Associations between stress and cycle outcomes (clinical pregnancy and indicators of oocyte and embryo quality) were measured by logistic or linear regression, adjusted for age, body mass index, education, smoking, alcohol and caffeine consumption, shiftwork and night work. RESULTS: Ultrasound verified pregnancy rate was 26.6% overall per cycle started and 32.9% per embryo transfer. Stress measures were not associated with clinical pregnancy: when compared with the lowest categories, the adjusted odds ratio (OR) and 95% confidence interval (CI) for the highest categories of the perceived stress score was 1.04 (95% CI 0.58-1.87), infertility-related stress score was OR = 1.18 (95% CI 0.56-2.47), morning and evening cortisol was OR = 1.18 (95% CI 0.60-2.29) and OR = 0.66 (95% CI 0.34-1.30), respectively. CONCLUSIONS: Perceived stress, infertility-related stress, and cortisol levels were not associated with IVF cycle outcomes. These findings are potentially reassuring to women undergoing fertility treatment with concerns about the influence of stress on their treatment outcome.

Effects of Fluoxetine on Human Embryo Development.

The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 µM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 µM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 µM, but not 0.25 µM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment.

Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development.

UNLABELLED: Histidine-rich glycoprotein (HRG) is an abundant plasma protein involved in multiple biological processes including immunology, vascularisation, and coagulation. These processes are of importance in regulating embryo development and implantation. A specific polymorphism in the HRG gene, HRG C633T, has an impact on various aspects of fertility, such as oocyte quality, endometrial receptivity, and possibly the capacity of the embryo itself to implant. To further examine the potential role of the HRG C633T polymorphism in regulating endometrial angiogenesis and on embryo development, two HRG peptides were constructed. These HRG peptides correspond to the amino acids 169-203 of the protein which, in turn, reflects the C633T polymorphism in the gene. The HRG proline or serine peptides were added to cultures of primary human endometrial endothelial (HEE) cells and to human embryos in vitro. The HRG peptides inhibited vascular endothelial growth factor (VEGF) induced proliferation and migration and promoted tube formation of HEE cells. The embryos were monitored using a time-lapse system (EmbryoScope®). Except for a prolonged time from first cleavage after thawing to development of the morula, no difference in embryo morphokinetics or embryo quality was noted in human embryos cultured in the presence of the HRG proline peptide. Taken together, these results suggest that treatment with a specific HRG peptide might prime the endometrium for implantation and be beneficial for adequate placentation. However, addition of a specific HRG proline peptide to human embryos has no beneficial effects in terms of embryo development. ABBREVIATIONS: HRG: histidine-rich glycoprotein; HEE: human endometrial endothelial; VEGF: vascular endothelial growth factor; TSP: thrombospondin; SNP; single nucleotide polymorphism; IVF: in vitro fertilization; CLESH-1: CD36 LIMPII Emp structural homology domain-1; ECM: endothelial cell medium; FBS: fetal bovine serum; cDNA: complementary DNA.

Exploratory study of the association of steroid profiles in stimulated ovarian follicular fluid with outcomes of IVF treatment.

Steroid concentrations in stimulated follicular fluid (sFF) samples have been linked to the quality of oocytes used in IVF treatments. Most of the published studies focused on evaluating the association of the IVF outcomes with only a few of the steroids, measured by immunoassays (IA). We performed a treatment outcome, prospective cohort study using stimulated FF sampled from 14 infertile women undergoing IVF treatment; single oocyte was used per IVF cycle. Fourteen endogenous steroids were analyzed in 22 ovarian follicle aspirations, which corresponded to the embryos used in the IVF. Ten oocytes were associated with live birth (LB) and 12 with no pregnancy (NP). Steroids were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Differences in distribution of concentrations in association with the pregnancy outcome (LB or NP), and receiver operating characteristic (ROC) curves analysis were performed for the entire cohort and for within-women data. The predominant androgen and estrogen in stimulated sFF were androstenedione (A4) and estradiol (E2), respectively. Lower concentrations of pregnenolone (Pr), lower ratios of A4/ dehydroepiandrosterone (DHEA), testosterone (Te)/DHEA, and greater ratios of E2/Te, and estrone/A4 were observed in sFF samples associated with LB. Among the oocytes associated with NP, in four out of 12 samples total concentration of androgens was above the distribution of the concentrations in the oocytes corresponding to the LB group. Observations of the study indicated increased consumption of precursors and increased biosynthesis of estrogens in the follicles associated with LB. Our data suggest that potentially steroid profiles in sFF obtained during oocyte retrieval may serve as biomarkers for selection of the best embryo to transfer after IVF.

Physical activity, fatness, educational level and snuff consumption as determinants of semen quality: findings of the ActiART study.

In this study, the association between physical activity and other potential determinants, objectively measured by accelerometry, was examined. Sixty-two men attending an infertility clinic participated in the study. Obese men (body mass index ≥ 30) and those with a waist circumference 102 cm or more had lower semen volume than the other men (P < 0.05). Higher values in sperm parameters were observed in participants who completed university studies and those who did not consume snuff, compared with the other participants (P < 0.05). Finally, men who spent an average number of 10 min-bouts of moderate-to-vigorous physical activity had significantly better semen quality than those who engaged in low or high numbers of bouts of activity (P < 0.05). No associations were found for sedentary or moderate-to-vigorous physical activity time when it was not sustained over 10 min, i.e. not in bouts. Men who have average levels of physical activity over sustained periods of 10 min are likely to have better semen quality than men who engage in low or high levels of such activity. Similarly, high levels of total and central adiposity, low educational level and snuff consumption are negatively related to semen quality.

Full-term newborn after repeated ovarian tissue transplants in a patient treated for Ewing sarcoma by sterilizing pelvic irradiation and chemotherapy.

We report the first successful transplantation of cryopreserved ovarian cortical tissue into heavily irradiated tissues in a patient who had received sterilizing pelvic radiotherapy (54 Gy) and 40 weeks of intensive high-dose chemotherapy for the treatment of Ewing's sarcoma 14 years earlier. Repeated transplantation procedures were required to obtain fully functional follicular development. Enlargement of the transplants over time and increase of the size of the uterus were demonstrated on sequential ultrasonographic examinations. Eggs of good quality that could be fertilized in vitro were obtained only after a substantial incremental increase of the amount of ovarian tissue transplanted. Single embryo replacement resulted in a normal pregnancy and the birth of a healthy child by cesarean section at full-term. No neonatal or maternal postoperative complications occurred. Women facing high-dose pelvic radiotherapy should not be systematically excluded from fertility preservation options, as is currently the trend.

Glycoprotein 130 promotes human blastocyst development in vitro.

OBJECTIVE: To investigate the efficacy of leukemia inhibitory factor (LIF) and/or glycoprotein 130 (gp130) on in vitro growth of human embryos. DESIGN: Laboratory study. SETTING: University hospital-based IVF clinic. PATIENT(S): A total of 164 frozen embryos that survived thawing were cultured in media supplemented with LIF and/or gp130 or control media. INTERVENTION(S): Morphological development was evaluated by light microscopy. Protein expression profiles of single blastocysts were evaluated using matrix-assisted laser desorption/ionization time of flight-based intact cell mass spectrometry. MAIN OUTCOME MEASURE(S): Embryo development and protein content. RESULT(S): Addition of gp130 to culture media improved blastocyst formation (73% vs. 43%). Addition of LIF to the culture media did not improve embryo development. Protein fingerprint spectra were obtained that revealed significant intensity changes for multiple molecular species including thymosin beta-10, thymosin beta-4, histone H2A, histone H2B, histone H4, ubiquitin, ubiquitin-T, and acyl-CoA binding protein. CONCLUSION(S): Glycoprotein 130, but not LIF, seems to be beneficial for preimplantation embryo development, implicating a physiological role in regulating preimplantation development in humans and thus ought to be included in culture media designed for embryo culture to the blastocyst stage. Furthermore, these findings highlight the great potential of matrix-assisted laser desorption/ionization time of flight mass spectrometry and intact cell mass spectrometry as a versatile tool in reproductive medicine research.

Proteomic analysis of human blastocoel fluid and blastocyst cells.

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required on a large scale. In the undifferentiated state, hESCs appear to possess an unlimited potential for proliferation, but optimal, defined, and safe culture conditions remain a challenge. The aim of the present study was to identify proteins in the natural environment of undifferentiated hESCs, namely, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass spectrometry, 286 proteins were identified in the blastocoel fluid and 1,307 proteins in the corresponding cells of the blastocyst. Forty-two were previously uncharacterized proteins-8 of these originated from the blastocoel fluid. Furthermore, several heat shock proteins (Hsp27, Hsp60, Hsc70, and Hsp90) were identified in blastocoel fluid together with zona pellucida proteins (ZP2-4), Vitamin D-binding protein, and Retinol-binding protein 4. Proteins that regulate ciliary assembly and function were also identified, including Bardet-Biedl syndrome protein 7. This study has identified numerous proteins that cells from the ICM of the human blastocyst are exposed to via the blastocoel fluid. These results can be an inspiration for the development of improved culture conditions for hESCs.

N-glycans of human protein C inhibitor: tissue-specific expression and function.

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x). A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⁻¹ s⁻¹ for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⁻¹ s⁻¹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

Prostasomal DNA characterization and transfer into human sperm.

Human prostasomes, exosome-like microvesicles secreted by acinar cells of the prostate gland, contain chromosomal DNA. Agarose gel electrophoresis of DNA from seminal prostasomes displayed fragments of over 12 kb and smaller, with a distinct band around 1 kb that was excised, cloned, and sequenced. The sequences showed 8 out of 25 clones (32%) originating from genes. We elaborated the concept further by carrying out a genome-wide DNA copy number analysis of prostasomal DNA, hypothesizing that human prostasomes contain fragments of DNA randomly selected from the entire genome. Acridine orange-stained prostasomes were incubated with freshly prepared sperm for different times, and a transfer of acridine orange-stained prostasomal DNA to sperm (preferentially the head region) was observed. Fluorescence microscopy of slices in the center of 14 optical slides of the sperm head displayed an even fluorescence rather than a halo-like one, indicating DNA-uptake rather than just binding along the sperm head membrane.

Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue.

BACKGROUND: Controlled-rate freezing of ovarian cortical tissue for preservation of fertility among young women facing chemo- or radio-therapy is a widely accepted procedure. To improve the method for cryopreservation of ovarian tissue, particularly the stroma, we carried out a systematic comparison of vitrification versus slow programmed freezing. METHODS: Ovarian tissue from 20 women, donated during Caesarean section, was used for parallel comparison of survival and detailed light and electron microscopic (EM) morphology of oocytes, granulosa cells and ovarian stroma after freezing (slow freezing and vitrification), thawing and 24-h culture. Using tissue obtained from the same patient, we compared four cryopreservation protocols and fresh tissue. The cryoprotectants used in slow freezing were 1,2-propanediol (PrOH)-sucrose and ethylene glycol (EG)-sucrose. For vitrification, tissues were incubated for 5 or 10 min in three solutions containing a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP). RESULTS: Cryopreservation using controlled-rate freezing and vitrification preserved the morphological characteristics of ovarian tissue generally well. As revealed by morphological analysis, particularly EM, the ovarian stroma was significantly better preserved after vitrification than after slow freezing (P < 0.001). The follicles were similarly preserved after all freezing methods. CONCLUSIONS: Vitrification using a combination of PrOH, EG, DMSO and PVP was comparable to slow freezing in terms of preserving follicles in human ovarian tissue. Ovarian stroma had significantly better morphological integrity after vitrification than after controlled-rate freezing.

Fertility preservation in girls with turner syndrome: prognostic signs of the presence of ovarian follicles.

CONTEXT: Many girls with Turner syndrome have follicles in their ovaries at adolescence. OBJECTIVE: Our objective was to study which girls might benefit from ovarian tissue freezing for fertility preservation. DESIGN: Clinical and laboratory parameters and ovarian follicle counts were analyzed among girls referred by 25 pediatric endocrinologists. SUBJECTS AND SETTING: Fifty-seven girls with Turner syndrome, aged 8-19.8 yr, were studied at a university hospital. INTERVENTIONS: Ovarian tissue was biopsied laparoscopically, studied for the presence of follicles, and cryopreserved. Blood samples were drawn for hormone measurements. MAIN OUTCOME MEASURES: Presence of follicles in the biopsied tissue related to age, signs of spontaneous puberty, karyotype, and serum concentrations of gonadotropins and anti-Müllerian hormone were assessed. RESULTS: Ovarian biopsy was feasible in 47 of the 57 girls. In 15 of the 57 girls (26%), there were follicles in the tissue piece analyzed histologically. Six of seven girls (86%) with mosaicism, six of 22 (27%) with structural chromosomal abnormalities, and three of 28 with karyotype 45X (10.7%) had follicles. Eight of the 13 girls (62%) with spontaneous menarche had follicles, and 11 of the 19 girls (58%) who had signs of spontaneous puberty had follicles. The age group 12-16 yr had the highest proportion of girls with follicles. Normal FSH and anti-Müllerian hormone concentrations for age and pubertal stage were more frequent in girls with follicles. CONCLUSIONS: Signs of spontaneous puberty, mosaicism, and normal hormone concentrations were positive and statistically significant but not exclusive prognostic factors as regards finding follicles.