Oocyte Collection Research Articles

Outcomes of random start versus clomiphene citrate and gonadotropin cycles in occult premature ovarian insufficiency patients, refusing oocyte donation: a retrospective cohort study

The aim of this study is to present the clinical outcomes of a random start, a spontaneous folliculogenesis protocol versus Clomiphene Citrate and Gonadotropin treatment in women with occult premature ovarian insufficiency. Women underwent treatment between 1 February 2009, and 30 May 2016. 41 women were treated with the random start protocol while 48 cases received ovarian stimulation with clomiphene and gonadotropins. All included cases met the criteria of 4 months of oligo-ovulation, follicular-stimulating hormone levels over 30 IU/L and anti-Mullerian hormone levels below 0.30 ng/mL. The random start protocol involved following the subjects for up to 6 months until spontaneous folliculogenesis occurred. The mean number of oocytes collected, mature oocytes, fertilized oocytes, and grade II embryos were significantly higher in the random start protocol (p < .05). The doses of gonadotropin administration and hCG were significantly lower in the random start protocol (p < .05). The clinical pregnancy and live birth rates were significantly higher in the random start protocol (p < .05). Likely stimulation is of little benefit in women with occult premature ovarian insufficiency. Observation while waiting for spontaneous folliculogenesis results in better outcomes, and less oocyte collections.

Retrievable hydrogels for ovarian follicle transplantation and oocyte collection.

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two-cell (36%), and four-cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer-laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients withcancer.

Outcome of immature oocytes collection of 119 cancer patients during ovarian tissue harvesting for fertility preservation.

Few clinical options for fertility preservation are available to females with cancer, and data about clinical outcomes is limited. Potential supplementary approaches to fertility preservation include retrieval of immature oocytes followed by in vitro maturation (IVM) and storage. The aim of this study was to evaluate post-thawing outcomes of immature oocytes collected both by transvaginal aspiration and from excised ovarian tissue. We conducted a retrospective cohort study of patients treated in a single tertiary center. We reviewed the records of 119 cancer patients who underwent ovarian tissue cryopreservation and immature oocyte harvesting for fertility preservation. All embryos and oocytes that were frozen and thawed were included in the study. Post-thawing outcomes were evaluated. Thirty-five stored embryos from eight patients were thawed. Twenty-nine embryos survived (82% survival rate) and were transferred. Six oocytes were thawed, two oocytes survived, and no oocytes were fertilized. Only one PCOS patient became pregnant, resulting in the normal delivery of a healthy baby. Although a relatively high number of mature oocytes and embryos can be stored with the combined procedure, the limited rate of pregnancies represents a poor reproductive outcome. Therefore, this approach should be reserved for special groups with limited options.

Response: Commentary: Efficacy of Follicle-Stimulating Hormone (FSH) Alone, FSH + Luteinizing Hormone, Human Menopausal Gonadotropin or FSH + Human Chorionic Gonadotropin on Assisted Reproductive Technology Outcomes in the "Personalized" Medicine Era: A Meta-analysis.

Setting: Luteinizing hormone(LH) and human chorionic gonadotropin (hCG) act on the same receptor, activating different signal transduction pathways. The role of LH or hCG addition to follicle stimulating hormone (FSH) as well as menopausal gonadotropins (hMG) in controlled ovarian stimulation (COS) is debated. Objective: To compare FSH+LH, or FSH+hCG or hMG vs FSH alone on COS outcomes. Design: A meta-analysis according to PRISMA statement and Cochrane Collaboration was performed, including prospective, controlled clinical trials published until July 2016, enrolling women treated with FSH alone or combined with other gonadotropins. Trials enrolling women with polycystic ovarian syndrome were excluded (PROSPERO registration n. CRD42016048404). Results: Considering 70 studies, the administration of FSH alone resulted in higher number of oocytes retrieved than FSH+LH or hMG. The MII oocytes number did not change when FSH alone was compared to FSH+LH, FSH+hCG or hMG. Embryo number and implantation rate were higher when hMG was used instead of FSH alone. Pregnancy rate was significantly higher in FSH+LH-treated group versus others. Only twelve studies reported live birth rate, not providing protocol-dependent differences. Patients’ stratification by GnRH agonist/antagonist identified patient subgroups benefiting from specific drug combinations. Conclusion: In COS, FSH alone results in higher oocyte number. HMG improves the collection of mature oocytes, embryos and increases implantation rate. On the other hand, LH addition leads to higher pregnancy rate. This study supports the concept of a different clinical action of gonadotropins in COS, reflecting previous in vitro data.

Utilization of Ovary Collected from Abattoir as Genetic Material Resources

&lt;p&gt;Slaughtering productive cow is commonly practiced, eventhough it is not in accordance with government program to increase livestock population in Indonesia. Ovarium from slaughtered cow contains genetic materials that have not utilized properly. The purpose of this paper is to review ovarian utilization collected from abattoir through &lt;em&gt;in vitro&lt;/em&gt; fertilization technology. The best procedure of ovary preservation from abbatoir to the laboratory, oocyte collection techniques are required to produce qualified embryo. Transportation of ovary to laboratory requires proper temperature and time. Slicing method obtains high quantity and good quality of oocyte. The quality of oocytes collected from abattoir as good as those taken from live animal. Ovaries that previously as waste product can be used for &lt;em&gt;in vitro&lt;/em&gt; production of embryos and livestock genetic material resources.&lt;/p&gt;

Developmental competence of cat (Felis domesticus) oocytes and embryos after parthenogenetic stimulation using different methods.

The aim of this study was to compare the effects of various activating factors on feline oocytes. The study included activation within the ovary (natural), activation during in vitro maturation (spontaneous activation), chemical activation (ionomycin + 6-DMAP), activation by spermatozoa and injection (ICSI) and mechanical activation (sham ICSI). According to our results, parthenogenetic embryos could emerge at every step of in vitro embryo production (IVP) procedures. After oocyte collection, 6% of parthenogenetic embryos were observed, mainly at the 2-4-blastomere stages. After 24 h of in vitro maturation, parthenogenetic activation was observed in 7% of oocytes. Using ionomycin and 6-DMAP to artificially activate oocytes, 53% of cleaved embryos were obtained. The results after ICSI (54% cleaved embryos) were not significantly different from the results in Group III using chemical activation (53% cleaved embryos). But only after ICSI were blastocysts obtained (5/73.7%) as a result of in vitro culture. Moreover, embryos after ICSI were of the best morphological quality with minor levels of fragmentation evident in the embryos. After sham mechanical activation, 'sham ICSI', 8% of cleaved embryos were noted. Therefore, it is advised to maintain a negative control in parallel with each step of IVP techniques, to avoid misleading results. Chemical methods for artificial activation of feline oocytes are the most promising for application to the cloning and production of parthenogenetic embryos for experimental studies.

In vivo ultrasound guided transvaginal oocyte collection in the cow

This paper describes the results of the in vivo ultrasound guided follicular aspiration for ovum pick υρ (OPU) in the cow. Twelve non pregnant dry cows aged 4-6 years were used in this experiment. Eight cows underwent OPU during three successive oestrous cycles and another four cows were used as controls having only transvaginal ultrasound scanning of their ovaries. Oocyte collection took place three times during the luteal phase of each natural oestrous cycle (days 3-4,9-11 and 14-17). The content of 326 follicles with a diameter of 4-15mm was aspirated and 104 oocytes were collected (recovery rate 31.9% or 1.55 oocytes per cow and session). The oocyte recovery rate increased after the first three sessions (from 13.04% to 35.0%) and reached levels of υρ to 52.6%. More follicles were aspirated on days 9-11 (133 follicles 40.8%) compared to 111 (34%) follicles on days 14-17 and 82 (25%) on days 3-4) (P&lt;0.05). The evaluation of the collected oocytes revealed that 60 oocytes (57.7%) were suitable for further in vitro manipulation. Neither the origin of the oocyte (left or right ovary) nor the stage of the oestrous cycle affected the recovery rate or the quality of the collected oocytes. There was no significant difference either in the length of the oestrous cycle between the experimental animals and the controls (21.6± 1.4 vs. 22.37±1.0 respectively), or in plasma progesterone concentration in daily collected blood samples from the animals of the two groups. The results of this study are compared to those from the international literature and to the results from endoscopical methods for oocyte recovery. The feasibility of application of this technique to projects designed to improve the genetic merit of cows is discussed.

ЕФЕКТИВНІСТЬ ЗАСТОСУВАННЯ ПРЕГРАВІДАРНОЇ ПІДГОТОВКИ У ПАЦІЄНТОК ІЗ СИНДРОМОМ ПОЛІКІСТОЗНИХ ЯЄЧНИКІВ В ПРОГРАМАХ ДОПОМІЖНИХ РЕПРОДУКТИВНИХ ТЕХНОЛОГІЙ ПРИ ЗАСТОСУВАННІ ПРОТОКОЛІВ КОНТРОЛЬОВАНОЇ ОВАРІАЛЬНОЇ СТИМУЛЯЦІЇ З АНТАГОНІСТАМИ ТА ТРИГЕРОМ АГОНІСТОМ

Синдром полікістозних яєчників (СПКЯ) – полісиндромне захворювання, що часто асоціюється з безпліддям, і є найчастішою патологією у жінок репродуктивного віку як в Україні, так і за кордоном. За даними літератури, СПКЯ діагностують майже у 50 % жінок із порушеннями менструальної та овуляторної функцій, та у 40 % пацієнток, які страждають від ендокринного безпліддя.Мета дослідження – оцінити ефективність прегравідарної терапії перед проведенням контрольованої стимуляції овуляції в протоколах ЕКЗ у пацієнток із СПКЯ.Матеріали і методи. Рандомізоване клініко-лабораторне дослідження було проведено серед 146 жінок із верифікованим діагнозом СПКЯ та наступними характеристиками: вік 29,8 (25–38) років, ІМТ – (27,2±4,9) кг/м², АМГ – 6,3 ng/ml (4,1–11,6 ng/ml). Тривалість безпліддя – від 2 до 19 років у середньому (6,8±0,5) року. Первинне безпліддя було у 102 пацієнток (69,9 %), а вторинне – в 44 жінок (30,1 %). Середній вік жінок, у яких починалось менархе, складав (14,1±0,3) року, в усіх жінок цикл був не регулярним, у 118 жінок (80,8 %) менструальний цикл тривав від 30 до 150 днів. Обстеження та лікування проводили відповідно до наказу Міністерства охорони здоров’я України № 787 від 09.09.2013р. “Про затвердження Порядку застосування допоміжних репродуктивних технологій в Україні” та наказу Міністерства охорони здоров’я України від 29.11.2013р. № 1030/102 “Про удосконалення системи планування сім’ї та охорони репродуктивного здоров’я в Україні”. Перед програмою ДРТ було проведено збір даних щодо попереднього обстеження та лікування подружніх пар із подальшим обов’язковим стандартним протоколом обстеження (ОСПО).Результати досліджень та їх обговорення. У статті показано ефективність терапії за допомогою препаратів “FT-500 Plus” (інозитол, фолієва кислота, глутатіон, лютеїн, цинк, вітамін С, вітамін Е, селен) та “Аквадетрин” вітаміну D3 в пацієнток із безпліддям при синдромі полікістозних яєчників (СПКЯ) перед проведенням контрольованої стимуляції овуляції (КОС) в протоколі екстракорпорального запліднення. Стимуляцію здійснювали при рекомбінантному гонадотропіні корифолітропіні-альфа “ЕЛ ОНВА” на 5–7 дні після протоколу залежно від реакції яєчників. Коли розмір фолікула досягав 14–15 мм, призначали щоденно антагоніст ГнРГ “Оргалутран” по 0,25 мг. На 8 день від введення препарату “ЕЛ ОНВА” продовжили стимуляцію рекомбінантним ФСГ “Пурегон” до кінцевого дозрівання ооцитів (3 фолікули&gt;17 мм), в якості тригера використовували агоніст релізинг-гормону “Диферелін”. Забір ооцитів здійснювали через 36 год від введення тригера овуляції із подальшою вітрифікацією ембріонів на 5–6 доби (стадія бластоцисти).Висновки. Отримані результати показали, що у пацієнток, які отримували прегравідарну терапію, сумарний коефіцієнт ефективності отриманих ооцитів, ступінь їх зрілості, частота запліднення, дроблення, вихід бластоцист та, відповідно, настання вагітності були вищими порівняно з контрольною групою.

Preventing Common Hereditary Disorders through Time-Separated Twinning

Biomedical advances have led to a relaxation of natural selection in the human population of developed countries. In the absence of strong purifying selection, spontaneous and frequently deleterious mutations tend to accumulate in the human genome and gradually increase the genetic load; that is, the frequency of potentially lethal genes in the gene pool. Gradual increase in incidence of many complex disorders suggests deleterious impact of the genetic load on human well-being. Recent advances in in vitro fertilization (IVF) combined with artificial twinning and transgenerational embryo cryoconservation offer the possibility of preventing significant accumulation of genetic load and reducing the incidence of hereditary disorders. Many complex diseases such as type 1 and 2 diabetes, autism, bipolar disorder, allergies, Alzheimer’s disease, and some cancers show significantly higher concordance in monozygotic (MZ) twins than in fraternal twins (dizygotic, DZ) or parent-child pairs, suggesting their etiology is strongly influenced by genetics. Preventing these diseases based on genetic data alone is frequently impossible due to the complex interplay between genetic and environmental factors. We hypothesize that the incidence of complex diseases could be significantly reduced in the future through a strategy based on time-separated twinning. This strategy involves the collection and fertilization of human oocytes followed by several rounds of artificial twinning. If preimplantation genetic screening (PGS) reports no aneuploidy or known Mendelian disorders, one of the MZ siblings would be implanted and the remaining embryos cryoconserved. Once the good health of the adult MZ sibling(s) is established, subsequent parenthood with the cryoconserved co-twins could substantially lower the incidence of hereditary disorders with complex etiology and virtually eradicate simple Mendelian disorders. The proposed method of artificial twinning has the potential to alleviate suffering and reduce the negative social impact induced by dysgenic effects associated with known and unknown genetic factors. Time-separated twinning has the capacity to prevent further accumulation of the genetic load and to provide source of isogenic embryonic stem cells for future regenerative therapies.

In vitro fertilization embryo transfer processes and pathway: A review from practice perspective

Infertility affects approximately 10%–15% of reproductive-aged couples. In Nigeria, it is an important reproductive health issue and a common reason for gynecological clinic consultations. Significant improvements in fertility treatment have made it possible for many patients to conceive with medical assistance. For example, women with fallopian tube blockage can conceive with in vitro fertilization (IVF). Modern IVF generally involves controlled ovarian stimulation with exogenous gonadotropins, oocytes collection through transvaginal ultrasonographic-guided aspiration, coculture of eggs and sperm in culture (or intracytoplasmic injection of sperm into the oocyte), and placement of the resultant embryos (2–5 d later) directly into the uterus. Some of the major drawbacks to IVF are its high cost of treatment and paucity of availability in our sub-Saharan region.

Missed estradiol determination resulting in oocyte retrieval and embryo development following controlled ovarian hyperstimulation at early pregnancy: Case report

This paper is a case report on the success of oocyte retrieval and good quality embryo development following controlled ovarian hyperstimulation at early pregnancy. A 30-year-old patient underwent controlled ovarian hyperstimulation by gonadotropin-releasing hormone agonist long protocol. On the day of oocyte collection, a 5-week gestational sac was observed by exact sonography monitoring. However, via ultrasound guided follicle puncture, 7 oocytes were collected. After intarcytoplasmic sperm injection, 3 developed good quality embryos were cryopreserved. Moreover, the natural pregnancy was continued and finally a healthy live birth was achieved. Despite physiological hormonal changes during pregnancy, the follicular growth occurred and followed by oocyte retrieval and embryo development, subsequently.

158 The Effect of Follicular Wave Phase at Time of Ovum Pick-Up on Bovine Oocyte Cytoplasmic Maturation and Developmental Competence

During oocyte collection, follicular wave phase is unknown, although differences in follicle environment may have dramatic effects on oocyte quality. This project was performed to determine whether oocyte collection during different phases of the follicular wave affects oocyte competence. Oocytes were collected via transvaginal ultrasound guided oocyte aspiration from 18 cows, at 4, 8, and 12 days following dominant follicle removal, representing follicle wave emergence, peak, and atresia, respectively (160, 314, and 273 oocytes, respectively). Once recovered, oocytes were graded and assigned to either being held as immature or matured in vitro for 24 h. Oocytes were then stained in Mitotracker deep red, fixed and stained with an anti-IP3R1 primary antibody and an Alexa Fluor 488-conjugated secondary antibody, before being stained with DAPI, to identify mitochondria, inositol triphosphate receptor 1 (IP3R1), and chromatin respectively. Mitochondria were analysed based on cytoplasmic distribution and classified as peripheral (immature), diffuse, central (mature), or sparse. Expression of IP3R1 was measured as corrected total cell fluorescence in Image J (National Institutes of Health, Bethesda, MD, USA). Staining patterns were analysed using ANOVA. A subset of the matured oocytes was stained with Fluo-3 to measure cytoplasmic calcium levels. These oocytes were then parthenogenetically activated before being imaged again to view changes in calcium levels, and presumptive embryos were cultured for 4 days. Fluo staining was measured as intensity levels (none, slight, moderate, high) and differences in development and stain levels were analysed using the Kruskal-Wallis test. Although mitochondria location was unaffected by collection day, it was significantly affected by maturation status (P = 0.0036). However, oocytes showed incomplete mitochondrial maturation, with mitochondria residing in the diffuse orientation in the majority of oocytes. Expression of IP3R1 appeared to be more sensitive to treatment. Expression significantly increased as meiosis proceeded (P = 0.0081) and there was a significant difference in expression between oocyte collection days (P = 0.0026). The interaction between collection day and maturation status also had a significant effect (P = 0.048), with mature oocytes showing an increase in IP3R1 expression, most notable in those collected on Day 4. Oocyte quality had a notable effect on the ability of oocytes to progress through meiosis (P = 0.054) and on mitochondrial location (P = 0.053), with AB oocytes showing better maturation parameters in both respects. Although the day of collection did not affect embryo development, Fluo stain intensity was an indicator of embryo developmental potential (P = 0.053), with oocytes having decreased potential to develop if the initial calcium levels were moderate to high. Results suggest that oocyte collection during wave emergence yields a slight advantage in oocyte quality. Although IP3R1, necessary for Ca2+ spikes during fertilization, indicates competence, high levels of cytoplasmic Ca2+ at the time of activation appear to be detrimental to embryo development.

197 The Effect of Gonadotropin-Releasing Hormone Agonist on Superovulation and Estrous Synchronization in Female Sprague Dawley Rat

A large number of oocytes and adequate recipient conditions are required to produce genetically modified rats by embryo transfer. In this study, we investigated the effect of a synthetic gonadotropin-releasing hormone (GnRH) agonist, buserelin, on (1) superovulatory response in donor rats, and (2) oestrous cycle synchrony in recipient female Sprague Dawley rats at 8 weeks of age. First, for superovulation, donors were divided into 2 groups: control and buserelin injection group (42 μg mL−1). Superovulation was performed in both groups by injection of pregnant mare’s serum gonadotropin (PMSG, 150 IU) at Day −3 and human chorionic gonadotropin (hCG, 150 IU) at Day −1 (Day 0 = oocyte collection). In addition, the buserelin group was given 42 μg mL−1 buserelin on Day −4. Oviduct and ampulla were removed surgically to collect the oocytes. As results, more ovulations were detected in the 42 μg mL−1 buserelin group (6.18 ± 1.10 oocyte/rat, n = 240) than in the control group (5.06 ± 0.82 oocyte/rat; n = 124; t-test P = 0.038). Second, 2 groups were set up to investigate the effect of administration of buserelin on synchronizing the oestrus cycle of recipients. Because the oestrous cycle of the rat was 4 to 5 days, we examined the difference of vaginal impedance between the 2 groups on Day 4 after injection and checked whether there was a significant difference in the synchronization of the oestrous cycle. An impedance value &gt;3.0 kΩ was considered as an indication for proestrus stage; therefore, we analysed the data &gt;3.0 kΩ in both groups. In results, the incidence of proestrus was significantly increased in buserelin injection group (n = 128) than in the control group (n = 70). In addition, there was a significant difference in mean impedance in the 2 groups (buserelin injection group: 4.25 ± 3.79 kΩ; control group: 1.43 ± 1.20 kΩ; P &lt; 0.005). In conclusion, administration of 42 μg mL−1 buserelin had a beneficial effect on oocyte ovulation and oestrous synchronization, and it will be usefully applied in the production of transgenic rats.

4 Relationship Between Ovarian Vascularity, Cumulus–Oocyte Morphology and Luteal Development in Four-Month-Old Calves After FSH Stimulation

The objectives of this study were to determine the effect of LH on the blood flow to the ovaries of 4-month-old calves after 2 FSH stimulation protocols, and to examine the relationship between ovarian vascularity after superstimulation to the morphology of the cumulus–oocyte complexes (COC) and luteal function. We hypothesise that ovarian vascularity (detected by 3-dimensional (3D) analysis of Doppler ultrasound cineloops) will increase in response to LH, and the magnitude of change in vascularity would be predictive of (1) a greater proportion of expanded COC, (2) greater development of luteal tissue volume and vascularity at 3 and 7 days after follicle aspiration, and (3) higher levels of plasma progesterone. Ovarian superstimulation was initiated at the beginning of an induced follicular wave in 4-month-old beef calves (n = 16), and beef cattle &gt;16 months of age (control group, adults; n = 8) using either a traditional 4-day or an extended 7-day FSH protocol (n = 8 calves and n = 4 controls per group). Power Doppler ultrasound cineloops were recorded immediately before (i.e. 12 h after the last FSH treatment) and 24 h after LH treatment (before ultrasound-guided follicular aspiration for oocyte collection) to assess ovarian vascularity, and 3 and 7 days after follicular aspiration to assess luteal tissue volume and vascularity. Video segments were analysed in Fiji and Imaris software to obtain the 3D ovarian vascularity index (ratio of blood flow volume to tissue volume). The ovarian vascularity index tended to increase &gt;1.7-fold in response to exogenous LH in both prepubertal calves (pre-LH 1.5 ± 0.4% v. post-LH 2.6 ± 0.7%; P = 0.08) and adult cattle (pre-LH 2.2 ± 0.6% v. post-LH 4.7 ± 0.9%; P = 0.07). Calves with a recovery of &gt;75% of expanded COC had a higher ovarian vascularity index (10.7 ± 2.6% v. 4.8 ± 1.6%; P = 0.06) and luteal vascularity index (15.7 ± 4.5% v. 5.7 ± 2.1%; P &lt; 0.05) 7 days after aspiration than those with &lt;75% expanded COC. Calves in the 7-day FSH protocol had &gt;10-fold higher concentration of plasma progesterone on Day 3 (12.7 ± 7.3 ng mL−1 v. 1.2 ± 0.4 ng mL−1; P &lt; 0.05) and Day 5 (50.6 ± 28.0 ng mL−1 v. 4.5 ± 1.0 ng mL−1; P &lt; 0.05), and ~2-fold higher luteal vascularity index at 7 days after follicle aspiration (13.7 ± 4.6% v. 7.7 ± 2.8%; P &lt; 0.05) than calves in the 4-day FSH protocol, whereas no difference (P &gt; 0.05) was found in control (adult) animals. In conclusion, there was an increase in ovarian vascularity resulting from LH treatment in prepubertal calves and adult cattle. A greater proportion of expansion of COC at 24 h after LH treatment (an indicator of follicular maturation) was related to higher ovarian and luteal vascularity on Day 7 after collection in prepubertal calves, but not in adults. Luteal vascularity on Day 3 was reflective of plasma progesterone concentration, and prepubertal calves in the 7-day FSH protocol had greater plasma progesterone than calves in the 4-day FSH protocol. The use of FSH in calves allows a greater number of follicles for oocyte collection as it does in adult cattle. Research was supported by an NSERC grant.

Production, Preservation, and Transfer of South American Camelid Embryos.

The current review summarizes progress in the field of in vitro and in vivo production of South American Camelid embryos. Both methods require ovarian superstimulation (with FSH and eCG) to obtain multiple ovulations (in vivo embryo production) or to induce follicle growth for oocyte collection (in vitro embryo production). Moreover, superstimulation entails prior administration of hormones that inhibit follicular growth (progesterone, progestagens, and estrogens). Cumulus-oocyte complexes obtained must mature in vivo (buserelin administration) or in vitro to then be subjected to in vitro fertilization or intracytoplasmic sperm injection. All these techniques also require morphologically normal, motile spermatozoa to achieve fertilization. Methods used to decrease semen viscosity and to select the best spermatozoa (Percoll®; Androcoll-ETM) are described. Additionally, nuclear transfer or cloning has been applied in llamas. Up to now, embryo deep-freezing and vitrification have progressed slowly but are at the height of development. Embryos that are obtained by any of these techniques, either in vivo or in vitro, need to be transferred to synchronized recipient females. The best results are achieved after transfer to the left uterine horn with an ipsilateral ovulation. No live offspring have been obtained after the transfer of cryopreserved embryos. Applying reproductive biotechnologies, such as those described, will permit the expansion of genetically selected animals in the population and also that of wild camelid species, vicunas, and guanacos, whose embryos could then be transferred to the uterus of domestic species.

Antioxidants improve IVF outcome and subsequent embryo development in the mouse

What is the effect of a combination of three antioxidants (Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid), present in IVF medium during mouse oocyte and sperm collection, on fertilization and subsequent IVF embryo development? A combination of antioxidants resulted in faster developmental times from the 2-cell stage through to expanded blastocyst stage, accompanied by a significant increase in blastocyst cell number and a reduction of intracellular hydrogen peroxide (H2O2) levels. The antioxidant combination Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid, when present in embryo culture media, has a significant beneficial effect on in vitro fertilized mouse pronucleate oocyte development, especially under oxidative stress. IVF was conducted with combined antioxidants supplemented in IVF medium that was used for mouse oocyte collection and fertilization (oocyte IVF medium, 4 h exposure) and sperm collection and preparation (sperm IVF medium, 1 h exposure). IVF was conducted under 20% oxygen, in the presence or absence of a combination of antioxidants (10 μM Acetyl-L-Carnitine, 10 μM N-Acetyl-L-Cysteine, 5 μM α-Lipoic Acid) and resultant embryos cultured with and without antioxidants under 20% oxygen. Subsequently, the effects of antioxidants on either oocytes or sperm was evaluated. Embryo development was analysed through time-lapse microscopy followed by differential nuclear staining to determine cell allocation in the blastocyst. Intracellular levels of H2O2 were assessed using an aryl boronate probe after 4 h of incubation with antioxidants. Controls were gametes and embryos that had no antioxidants in the medium. In a separate series of experiments, pronucleate oocytes were collected in handling medium with and without antioxidants for 20 min and subsequent cell numbers analysed. Antioxidant treatment during both IVF and culture resulted in significantly faster development times to two cell cleavage (P < 0.01), which continued through to the expanded blastocyst stage (P < 0.05). Resultant blastocysts had a significant increase in both trophectoderm (TE) cell numbers, inner cell mass (ICM) and total cell numbers (P < 0.001). The addition of antioxidants to IVF medium or embryo culture media exclusively also resulted in a significant increase in both blastocyst TE and ICM numbers leading to an increase in total cell numbers (P < 0.001). Antioxidant supplementation of either oocyte IVF medium alone, or in both oocyte and sperm IVF medium, lead to significantly faster times to two cell cleavage, which continued through to the expanded blastocyst stage. Blastocyst cell number in both these groups had significantly higher TE cell numbers resulting in an increase in total cell numbers. In contrast, there were no differences in embryo developmental rates and blastocyst cell number when antioxidants were present only in the sperm IVF medium. Levels of H2O2 were significantly reduced in pronucleate oocytes that were cultured in the presence of antioxidants (P < 0.001) compared to control, untreated embryos. Similarly, pronucleate oocytes treated with the combined antioxidants during pronucleate oocyte collection resulted in significantly increased blastocyst ICM numbers compared with controls (P < 0.05). Embryo development was only examined in the mouse. These findings suggest that supplementation of antioxidants to the IVF medium, as well as to embryo culture media, may further assist in maintaining the viability of human embryos in ART, conceivably through the reduction of oxidative stress. This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

RADIATION EFFECT OF WIRELESS FIDELITY (Wi-Fi) ON OOCYTE NUMBER OF OOCYTE STIMULATION IN MICE (Mus Musculus)

Infertility is a problem experienced by some women and men around the world. Most infertility problems in women is caused by impaired reproductive organs or disrupted ovulation.. One factor that causes impaired oocyte maturation is wireless fidelity (Wi-Fi) radiation which has a radio frequency field of 2.45 GHz. The radiation may increase the activity of free radical cells through the fenton reaction pathways that cause infertility because of the disrupted oocyte development. This study aimed to determine the effect of Wi-Fi radiation on the number of oocytes. This was an experimental study using control group design. The subjects were 32 female mice selected through complete random sampling and divided into two groups: control (R0) and exposure (R1). Each group consisted of 16 mice. The Wi-Fi radio frequency used was 2.5 GHz. Mice (R1) were placed closer to Wi-Fi source (± 15cm), and there were two types of laptop PCs and 3G mobile phones connected to internet placed next to mice for 15 hours/28 days. The mice underwent a simultaneous cycle with intraperitoneal injection of PMSG and HCG. Furthermore, the mice were mated with vasectomized male monomatingly to induce ovulation. The fertilization pouch in both oviducts were observed for oocyte collection. The number of oocytes was calculated using an inverted microscope. There was a difference in the number of oocytes between control and exposure group. Statistical tests were analyzed using Mann Whitney U and resulted in significant values (p value = 0.00). No oocytes count in exposure group. In other words, the group underwent anovulation. In conclusion, Wi-Fi radiation affected the number of oocyte stimulation in mice. Therefore, it was important to minimize the risk factors that trigger electromagnetic radiation on reproductive health.

ID: 1087 Effects of oocytes collection and electro-activation protocols on maturation and preimplantation development of parthenogenetic diploid porcine embryos

Advances in porcine embryos production have been impaired by the in vitro maturation (IVM) system and in vitro developments (IVD) are still not optimized. The present study was undertaken to determine the effects of different oocytes collection methods (aspiration and dissection) on oocytes maturation and combine with the different electro-activation protocols on in vitro pre-implantation development of parthenogenetic diploid porcine embryos. The results showed that dissection method was significantly higher both in quantity (87.23%, p &lt;0.05) of matured oocytes and in the rate of oocytes with very good quality (53.00%) compared with aspiration method (62.00%, 30.22%; respectively). We further accessed the quality of matured oocytes competence via parthenogenetic embryo development. Using four electro-activation protocols, we found that the electro-activation condition had no effect on the development of embryos to the 2-cell, 4-cell and 8-cell stages. However, the oocytes activated by the 60V/cm amplitude of pulse group had less (41.33%; p &lt;0.05) development at the morula stage than 90V/cm, 100V/cm and 150V/cm amplitude of pulse groups (73.99%, 67.62%, 72.55%; respectively). The embryos develop to blastocyst stage was highest in the group induced by 90V/cm electro pulse was 71.76% compared to 100V/cm, 150V/cm, and 60V/cm (62.86%, 62.05%, and 37.63%, p&lt;0.001, respectively). We conclude that the combining of dissection method for porcine oocytes collection and electro-activation with the amplitude of pulses 90V/cm can enhance both the activation success and development competition in in vitro pre- implantation parthenogenetic diploid porcine embryos.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36–42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100–130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or in vitro-fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated Chk2-/- mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide-hydrate ("CHK2iII") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.