Effects of Pulsed-Wave Photobiomodulation Therapy on Human Spermatozoa.

BACKGROUND AND OBJECTIVES: Previous studies reported that photobiomodulation (PBM) positively affects the mitochondrial respiratory chain in sperm, resulting in improved motility and velocity. As laser settings are not yet fully established, the present study aimed at optimizing PBM on human sperm. In addition, possible side-effects of PBM on sperm DNA fragmentation level and acrosomal integrity have been analyzed. STUDY DESIGN/MATERIALS AND METHODS: A pulsed laser-probe (wavelength 655 nm, output power 25 mW/cm², impulse duration 200 nanoseconds) was used. Native fresh liquefied semen samples underwent radiation with energy doses of 0 (control), 4, 6, and 10 J/cm². Sperm parameters were assessed at 0, 30, 60, 90, and 120 minutes after radiation using a computer-assisted sperm analysis system. Motility and velocity of sperm from asthenozoospermic patients (n = 42) and normozoospermic controls (n = 22) were measured. The amount of DNA strand breaks was analyzed using ligation-mediated quantitative polymerase chain reaction in patients with asthenozoospermia (n = 18) and normozoospermia (n = 13). Post-irradiance acrosomal integrity was investigated using flow cytometry based on CD46 protein expression (n = 7). RESULTS: Exposure to laser energy-doses of 4 and 6 J/cm² improved sperm motility and velocity in asthenozoospermic patients. PBM exhibited no significant effect on DNA fragmentation level and expression of CD46 serving as a biomarker for acrosome integrity. CONCLUSION: PBM improves sperm motility parameters by maintaining DNA and acrosome integrity and, therefore, represents a promising new tool for assisted reproductive therapy. In particular, improving sperm motility in asthenozoospermic patients by PBM in future may contribute to increasing the chance for successful intrauterine insemination. The present trial has no clinical registration number, as only in vitro studies were performed. The study was approved by the local ethics committee and performed according to the Declaration of Helsinki. Lasers Surg. Med. © 2021 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Stratifying Cumulus Cell Samples Based on Molecular Profiling to Help Resolve Biomarker Discrepancies and to Predict Oocyte Developmental Competence.

To increase the efficiency of assisted reproductive techniques (ART), molecular studies have been performed to identify the best predictive biomarkers for selecting the most suitable germ cells for fertilization and the best embryo for intra-uterine transfer. However, across different studies, no universal markers have been found. In this study, we addressed this issue by generating gene expression and CpG methylation profiles of outer cumulus cells obtained during intra-cytoplasmic sperm injection (ICSI). We also studied the association of the generated genomic data with the clinical parameters (spindle presence, zona pellucida birefringence, pronuclear pattern, estrogen level, endometrium size and lead follicle size) and the pregnancy result. Our data highlighted the presence of several parameters that affect analysis, such as inter-individual differences, inter-treatment differences, and, above all, specific treatment protocol differences. When comparing the pregnancy outcome following the long protocol (GnRH agonist) of ovarian stimulation, we identified the single gene markers (NME6 and ASAP1, FDR < 5%) which were also correlated with endometrium size, upstream regulators (e.g., EIF2AK3, FSH, ATF4, MKNK1, and TP53) and several bio-functions related to cell death (apoptosis) and cellular growth and proliferation. In conclusion, our study highlighted the need to stratify samples that are very heterogeneous and to use pathway analysis as a more reliable and universal method for identifying markers that can predict oocyte development potential.

Assessment of reflectance confocal microscopy for non-invasive selection of optimal ovarian cortex fragments for autotransplantation.

RESEARCH QUESTION: Can reflectance confocal microscopy (RCM) be used to determine follicle density in human ovarian cortex fragments that are intended for fertility restoration? DESIGN: RCM was used on living cortex tissue fragments derived from five bovine ovaries and 13 human ovaries. All tissue fragments were cryopreserved and thawed before RCM analysis. Follicle numbers and distribution were determined by RCM and histology. Before and after RCM, general tissue viability and follicle integrity were assessed by a glucose uptake assay and neutral red staining, respectively. RESULTS: RCM can detect all stages of follicle development in living ovarian tissue to a maximum depth of 250 µm. In bovine tissue, all follicles were located within this 0-250 µm range. In human ovarian tissue, follicles were also present below the 250 µm RCM threshold, implying that only a percentage of the total number of follicles could be detected with RCM. The percentage of follicles detected by RCM appeared to be age dependent. The RCM procedure did not affect the glucose uptake by the tissue, whereas neutral red staining indicated a high level of follicle survival. CONCLUSION: In this proof of concept study, we have shown that RCM is a promising technique to determine the density of follicles ex vivo in living human ovarian cortex fragments, apparently without compromising the vitality of the tissue. Safety studies and further optimization of the RCM technique with a focus on increasing the penetration depth are required before clinical use of RCM.

Overnight ovarian tissue transportation for centralized cryobanking: a feasible option.

RESEARCH QUESTION: Is overnight transportation of ovarian tissue before cryopreservation in a centralized cryobank from the FertiPROTEKT network feasible? DESIGN: Data from 1810 women with cryopreserved ovarian tissue after overnight transportation from December 2000 to December 2017 were analysed with a focus on transportation, tissue activity parameters and pregnancy, and delivery rates after transplantation. RESULTS: A total of 92.4% of tissue samples arrived at ideal temperatures of 2-8°C, 0.4% were transported at temperatures lower than ideal and 6.4% were transported at temperatures that were too high, generally due to mishandling of the inlayed cool packs of the transportation boxes. In 62 women, 78 tissue transplantations were carried out. A subgroup of 30 women who underwent a single orthotopic transplantation with fulfilled criteria of a complete follow-up after transplantation until the end of study, a premature ovarian insufficiency after gonadotoxic therapy as well as the absence of pelvic radiation, was further analysed. In this group, transplantations into a peritoneal pocket accounted for 90%. Transplants were still active at 1 year and above after transplantation in 93.3%. Pregnancy and delivery rates were 46.7% and 43.3%, respectively, with one ongoing pregnancy at the end of the study. CONCLUSIONS: Overnight transportation for central cryobanking is a feasible concept that results in high reproducible success rates through standardized professional tissue freezing and storage.

Concept Paper on the Technique of Cryopreservation, Removal and Transplantation of Ovarian Tissue for Fertility Preservation.

The cryopreservation of ovarian tissue with subsequent transplantation of the tissue represents an established method of fertility protection for female patients who have to undergo gonadotoxic therapy. The procedure can be performed at any point in the cycle and thus generally does not lead to any delay in oncological therapy. With the aid of this procedure, more than 130 births to date worldwide have been able to be recorded. The birth rate is currently approximately 30% and it can be assumed that this will increase through the further optimisation of the cryopreservation and surgical technique. The concept paper presented here is intended to provide guidance for managing cryopreservation and transplantation of ovarian tissue to German-speaking reproductive medicine centres.

Fertility protection: complications of surgery and results of removal and transplantation of ovarian tissue.

Fertility-preserving measures are becoming important for patients receiving oncological treatment. One method involves cryopreservation of ovarian tissue and transplanting it when treatment is completed. We report complications resulting from surgical and fertility medicine, and the results of procedures for the removal and transplantation of ovarian tissue carried out within the FertiProtekt network. A survey using a structured questionnaire was conducted among the FertiProtekt network centres between November 2015 and June 2016. The analysis included surgical techniques used to remove and transplant ovarian tissue, surgical complications and results. Laparoscopic removal and transplantation of ovarian tissue have a low risk of complications. Surgical complications occurred in three of the network's 1373 ovarian tissue removals (n = 1302) and transplantations (n = 71); two complications (0.2%) occurred during removal and one during transplantation. Menstruation resumed in 47 out of 58 women (81%) who underwent ovarian tissue transplantation. Hormonal activity occurred in 63.2% of transplantations with a follow-up of 6 months or over. Sixteen pregnancies occurred in 14 patients, with nine births. The risks and complications of removal and transplantation of ovarian tissue are similar to those of standard laparoscopy. These procedures are becoming standard for fertility protection in cancer patients.

Xenotransplantation of cryopreserved human ovarian tissue--a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment.

OBJECTIVE: To systematically review the reporting of MII (MII) oocyte development after xenotransplantation of human ovarian tissue. DESIGN: Systematic review in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). SETTING: Not applicable. PATIENT(S): Not applicable. INTERVENTION(S): Formation of MII oocytes after xenotransplantation of human ovarian tissue. MAIN OUTCOME MEASURE(S): Any outcome reported in Pubmed. RESULT(S): Six publications were identified that report on formation of MII oocytes after xenotransplantation of human ovarian tissue. CONCLUSION(S): Xenografting of human ovarian tissue has proved to be a useful model for examining ovarian function and follicle development in vivo. With human follicles that have matured through xenografting, the possibility of cancer transmission and relapse can also be eliminated, because cancer cells are not able to penetrate the zona pellucida. The reported studies have demonstrated that xenografted ovarian tissue from a range of species, including humans, can produce antral follicles that contain mature (MII) oocytes, and it has been shown that mice oocytes have the potential to give rise to live young. Although some ethical questions remain unresolved, xenotransplantation may be a promising method for restoring fertility. This review furthermore describes the value of xenotransplantation as a tool in reproductive biology and discusses the ethical and potential safety issues regarding ovarian tissue xenotransplantation as a means of recovering fertility.

Spontaneous antral follicle formation and metaphase II oocyte from a non-stimulated prepubertal ovarian tissue xenotransplant.

BACKGROUND: Current strategies in cancer treatment have markedly increased the rates of remission and survival for cancer patients, but are often associated with subsequent sterility. While there are various options available to an adult female depending on the patient's particular situation, the only realistic option for preserving fertility in prepubertal females is to cryopreserve ovarian tissue. This is the first report of a morphologically mature oocyte collected from non-stimulated prepubertal ovarian tissue xenotransplants. METHODS: Ovarian tissue from a 6 year old patient suffering from nephroblastoma was removed and cryopreserved for fertility preservation. The frozen-thawed ovarian tissue fragments were xenotransplanted to bilaterally oophorectomized severe combined immunodeficiency (SCID) mice to assess follicle development. RESULTS: Antral follicle formation occurred post-xenotransplantation in a single ovarian fragment without exogenous hormone stimulation. A morphologically maturing oocyte was harvested from these follicles. CONCLUSIONS: Prepubertal human ovarian follicles and oocytes can be matured after xenotransplantation even without exogenous hormone stimulation. These results indicate that tissue collected from prepubertal patients can support fertility in cancer survivors.

The impact of culture conditions on early follicle recruitment and growth from human ovarian cortex biopsies in vitro.

OBJECTIVE: To investigate the effects of a dynamic fluidic culture system on early in vitro folliculogenesis in standardized ovarian cortex biopsies. DESIGN: Cortical small strips were cultured for 6 days in a conventional static or in a dynamic fluidic culture system. SETTING: University-affiliated laboratory with an associated cryobank facility. PATIENT(S): Ovarian cortex from postpuberal female cancer patients (26.1 ± 1.3 y) who opted for cryopreservation of their tissue for fertility protection before gonadotoxic cancer therapy. With informed consent of the Institutional Ethics Committee, part of the tissue was available for patient-related research studies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The viability and proliferative capacity of the cortex biopsies were evaluated by chemiluminescent microparticle immunoassay for detection of in vitro produced E2 and P in the supernate, by viable follicle counting via calcein staining, by histologic analyses, and by total RNA preparation and reverse transcription for real-time polymerase chain reaction of selected early folliculogenesis genes. RESULT(S): The data support the notion that early follicle development can be better achieved in vitro in a dynamic fluidic culture system. The findings are based on the presence of more viable follicles, higher expression levels of early folliculogenesis genes KIT-L, INHB, and GDF9, and the absence of premature luteinization of follicles. CONCLUSION(S): This study provides evidence that dynamic fluidic culture is a promising approach for investigating early follicular recruitment and growth in cortical biopsies. It may serve as a first step in a multistep culture system to design a complex in vitro system for complete folliculogenesis.

First live birth in germany after re-transplantation of cryopreserved ovarian tissue: original device for initiation of ice formation.

BACKGROUND: The problem of post-cancer infertility is of significant concern. The cryopreservation of ovarian tissue before cancer therapy with retransplantation after convalescence is the key to solving this problem. METHODS: Cryopreservation of ovarian tissue was performed in 2005 after surgical operation, post-operative low-temperature 22 hour transportation, and freezing using a special, original design block constructed for the initiation of ice formation (ice-seeding). We present the construction and function of this block. RESULTS: In 2011, it was noted that a baby was born after thawing and re-transplantation of ovarian tissue. The technical and biological aspects of initiated crystals formation in the process of cryopreservation are emphasised and discussed. CONCLUSIONS: The first live birth in Germany after re-transplantation of cryopreserved ovarian tissue was noted. This cryopreservation was performed using the protocol described here. Block for auto-seeding of principally new construction recommended.

Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility.

OBJECTIVE: To analyze whether a ready-to-use calcium ionophore improves outcomes, from fertilization to live birth, in patients with severe male factor infertility. DESIGN: Artificial oocyte activation offered to applicable patients over a 20-month period. SETTING: Specialized in vitro fertilization (IVF) centers in Austria and Germany. PATIENT(S): Twenty-nine azoospermic and 37 cryptozoospermic men. INTERVENTION(S): Mature oocytes treated with a ready-to-use Ca(2+)-ionophore (GM508 Cult-Active) immediately after intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Rates of fertilization, implantation, clinical pregnancy, and live birth. RESULT(S): Patients had had 88 previous cycles without artificial activation that resulted in a fertilization rate of 34.7%, 79 transfers (89.8%), and 5 pregnancies, which all spontaneously aborted except one. After artificial oocyte activation, the fertilization rate was 56.9%. In terms of fertilization rate, both azoospermic (64.4%) and cryptozoospermic (48.4%) men statistically significantly benefited from use of the ionophore. In 73 transfer cycles, positive ß-human chorionic gonadotropin levels were observed in 34 cases (46.6%) and 29 cycles (39.7%) that ended with a clinical pregnancy. The corresponding implantation rate was 33.3%. Four spontaneous abortions occurred (11.8%), and 32 healthy children were born. CONCLUSION(S): This is the first prospective multicenter study on artificial oocyte activation in severe male factor infertility. Present data indicate that a ready-to-use calcium ionophore can yield high fertilization and pregnancy rates for this particular subgroup. In addition to fertilization failure after ICSI, severe male factor infertility is an additional area for application of artificial oocyte activation.

The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle.

Following intracytoplasmic sperm injection (ICSI), some patients present low or zero fertilization rates. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. This study applied artificial oocyte activation in patient cohorts with a history of no fertilization (0%, group 1), fertilization between 1 and 29% (group 2) or fertilization between 30 and 50% (group 3) in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared with the previous cycle without activation. In group 1, fertilization rate was 41.6%, embryos for transfer were available in 82.1% of cycles, giving a clinical pregnancy rate of 18.8% and take-home baby rate of 12.8%. In group 2, despite a lower transfer rate (87.9% versus 100%, P<0.05), there were higher fertilization and clinical pregnancy rates (44.4% versus 19.3% and 31.4% versus 12.8%, respectively, P<0.05) and take-home baby rate was 24.1% versus 12.8%. In group 3, fertilization rates differed (56.1% versus 36.8%; P<0.001) but all other parameters were similar. Artificial oocyte activation has great potential especially in patients showing compromised fertilization rates below 30% after standard ICSI. Following intracytoplasmic sperm injection (ICSI), some patients present very low or even zero fertilization rates after ICSI. Artificial oocyte activation has been proposed as a suitable means to overcome this problem. We applied artificial oocyte activation in patients which presented a history either no fertilization, fertilization between 0 and 30% or fertilization between 30 and 50% in initial ICSI cycles. In the following treatment cycles, oocytes were activated after ICSI using a calcium ionophore. Fertilization, pregnancy and take-home baby rates were compared to the previous cycle without activation. For the groups with previously 0% or 1-29% fertilization, we noted higher fertilization rates and clinical pregnancy rates per embryo transfer. For the group with moderate fertilization, only fertilization rates differed but all other parameters were not significantly different. From these data we conclude that artificial oocyte activation has a great potential especially in patients which show a compromised fertilization rate below 30% in a standard ICSI cycle.

Retransplantation of cryopreserved ovarian tissue: the first live birth in Germany.

BACKGROUND: Cryopreserved ovarian tissue can be retransplanted to restore fertility after radiation or chemotherapy. To date, 15 live births after retransplantation have been reported worldwide. We report the first pregnancy and the first live birth after retransplantation in Germany. CASE REPORT: A 25-year-old female patient received initial chemotherapy and radiation of the mediastinum for Hodgkin's lymphoma in 2003 and suffered a relapse two years later. Ovarian tissue was laparoscopically removed and cryopreserved, and she was then treated with high-dose chemotherapy and stem cell transplantation. She remained in remission for 5 years and she could not conceive during this time. The cryopreserved ovarian tissue was thawed and laparoscopically retransplanted into a peritoneal pouch in the ovarian fossa of the right pelvic wall. Three months later, her menopausal symptoms resolved, and she had her first spontaneous menstruation. Six months after retransplantation, after two normal menstrual cycles, low-dose follicle stimulating hormone (FSH) treatment induced the appearance of a dominant follicle in the tissue graft. Ovulation was then induced with human chorionic gonadotropin (HCG), whereupon the patient conceived naturally. After an uncomplicated pregnancy, she bore a healthy child by Caesarean section on 10 October 2011. Histological examination of biopsy specimens revealed that the ovarian tissue of the graft contained follicles in various stages of development, while the original ovaries contained only structures without any reproductive potential. CONCLUSION: This was the first live birth after retransplantation of cryopreserved ovarian tissue in Germany and also the first case with histological confirmation that the oocyte from which the patient conceived could only have come from the retransplanted tissue. In general, young women who will be undergoing chemotherapy and/or radiotherapy for cancer must be informed and counseled about the available options for fertility preservation.

Live birth after ovarian tissue autotransplantation following overnight transportation before cryopreservation.

OBJECTIVE: To describe the first live birth after transplantation of ovarian tissue following overnight transportation of the tissue before freezing. DESIGN: Technical note. SETTING: University department of obstetrics and gynecology. PATIENT(S): A 25-year-old cancer survivor with previous Hodgkin disease and relapse. INTERVENTION(S): The ovarian tissue was kept cool for >20 hours in a special transport medium and a special cooling device before it was cryopreserved. After premature ovarian failure due to preconditioning chemotherapy for bone marrow transplantation, the cryopreserved ovarian tissue was transplanted orthotopically. MAIN OUTCOME MEASURE(S): Resumption of ovarian function after transplantation, recovery of fertility, and pregnancy. RESULT(S): Ovarian function returned in the patient 3 months after transplantation, as shown by follicle development and estrogen production. During the fifth menstrual cycle, mild stimulation with FSH was initiated in accordance with a low-dose protocol. When ultrasonography revealed a follicle 18-20 mm in size in the ovarian graft, hCG was added and the patient had sexual intercourse at the optimal time point. On day 14 of the luteal phase, hCG concentration and vaginal echography confirmed a viable intrauterine pregnancy, which resulted in a healthy live birth. CONCLUSION(S): Overnight transportation of ovarian tissue appears to be possible in combination with appropriate transportation logistics. However, further investigations are needed before this procedure can be offered as a chance for women to preserve fertility independently of direct access to a tissue-processing bank.

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results.

BACKGROUND: Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. METHODS: In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. RESULTS: Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1-15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. CONCLUSIONS: This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.

Xenotransplantation of cryopreserved ovarian tissue from patients with ovarian tumors into SCID mice--no evidence of malignant cell contamination.

This study examined the possible presence of malignant cells in ovarian cortex from patients with ovarian tumors after xenografting of the ovarian tissue into severe combined immunodeficiency mice. None of the mice presented symptoms of reintroduced malignancy nor did microscopic and immunohistochemical evaluation of the grafts raise any suspicion of residual malignant disease.

Gamete competence assessment by polarizing optics in assisted reproduction.

BACKGROUND: The purpose of this study was first to give an overview of the historical development of polarization microscopy, second to describe the various applications of this technique in assisted reproduction techniques (ART) and third to discuss the potential benefit of polarization microscopy as a predictor for IVF success. METHODS: The history of polarization microscopy was undertaken by performing a backward search in the scientific literature using Google and internet sites of several Societies for Microscopy and Cell Biology. Studies of polarization microscopy in ART were identified by using a systematic literature search in PubMed and Scopus. RESULTS: A total of 62 articles were identified by the direct search and further relevant articles were found by screening the cited literature in these articles. The topics relevant for assisted reproduction were spindle and zona imaging in combination with IVF success, meiotic cell cycle progression, pharmaceutical studies and cryopreservation. A separate topic was the use of sperm birefringence in ART. CONCLUSIONS: The majority of studies are observational studies and were not performed in a randomized manner and there is no direct comparison of techniques using other gamete selection markers. Despite this, most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis. Whether certain applications such as spindle or zona imaging may lead to an increase in IVF success is unclear at present. Publications on the use of polarization microscopy on sperm are still very limited.

Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles.

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor.

Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells.

The phthalate ester mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of di-(2-ethylhexyl) phthalate, a high-production-volume chemical used as a plasticizer and solvent in numerous consumer products. MEHP has been demonstrated to be a reproductive toxicant in rodents decreasing estradiol and progesterone production in preovulatory granulosa cells. In the present study, we examined the effect of MEHP on steroid production of human granulosa-lutein (GL) cells. Human GL cells collected from women undergoing in vitro fertilization were cultured in medium containing FSH, hCG and 8-Br-cAMP, respectively, together with various concentrations of MEHP (0-500 micromol L(-1)). After incubation for 48 h estradiol and progesterone were assayed in the spent culture medium. Furthermore, aromatase activity and mRNA levels of GL cells were determined. Basal as well as FSH-, hCG- and 8-Br-cAMP-stimulated estradiol production of GL cells was suppressed by MEHP in a dose-dependent manner (IC(50)=105 micromol L(-1), 138 micromol L(-1), 49 micromol L(-1) and 78 micromol L(-1)). Furthermore aromatase activity and mRNA levels were reduced in GL cells cultured with MEHP. In contrast, MEHP did not alter the production of progesterone up to a concentration of 167 micromol L(-1). The present data indicate that MEHP is a specific inhibitor of estradiol production in human GL cells with a post-cAMP site of action. The inhibition of estradiol production obviously results from a reduction of aromatase activity on the transcript level. As the in vitro effective doses of MEHP are within the range of real environmental exposure levels an inhibitory effect on estrogen production in vivo seems to be possible.

Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation.

This investigation compared conventional freezing of human ovarian tissue using either spontaneous or initiated ('seeded') ice formation. Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy. Small pieces of experimental tissue were randomly distributed into three groups that were then subjected to different treatments prior to culture in vitro for 16 days: the control group, no treatment, cultured immediately after biopsy (group 1); cryopreservation/thawing with spontaneous ice formation (group 2); and cryopreservation/thawing with initiated ice formation (group 3). Follicle viability and hormonal activity were then evaluated. There was no significant difference between groups regarding the concentration of oestradiol 17-beta in the culture supernatant, whereas progesterone concentration was significantly (P < 0.05) higher in group 1 compared with group 2 or 3. There was a significant (P < 0.05) difference in primordial and primary follicle density between all of the groups (group 1 having the highest and group 2 having the lowest) and group 2 had significantly (P < 0.05) fewer normal grade follicles than the other two groups. For optimal cryopreservation of human ovarian tissue, the protocol of conventional freezing should therefore include a step of initiated ice formation.