Evaluation of an automated dish preparation system for IVF and embryo culture using a mouse mode.

Manual dish preparation for IVF in human fertility clinics or animal laboratories heavily relies on embryologists' experience, which can lead to occupational illness due to long-term and monotonous operation. Therefore, introducing an automated technique to replace traditional methods is crucial for improving working efficiency and reducing work burden for embryologists. In the current study in the mouse, both manual and automated methods were used to prepare IVF or embryo culture dishes. A one-way analysis of variance was conducted to compare several factors, including preparation time, qualified rates, media osmolality of dishes, fertilization rates, and embryonic development to assess the efficiency and potential of automated preparation. The results showed that automation system significantly reduced the required time and increased the efficiencies and qualified rates of dish preparation, especially for embryo culture dishes, without significantly altering medium osmolalities. There were no significant differences between two preparations in fertilization rates and embryo development in mice. Thus, automated dish preparation can improve working efficiency and qualified rates while maintaining fertilization rates and subsequent embryonic development without compromising osmolality stability of medium. It presents a superior alternative to manual preparation, reducing the workload of embryologists and facilitating the standardization of operational procedures.

Automation in vitrification and thawing of mouse oocytes and embryos.

Vitrification is a common technique for cryopreserving oocytes or embryos. However, manual vitrification is tedious and labor-intensive, and can be subject to variations caused by human factors. To address these challenges, we developed an automated vitrification-thawing system (AVTS) based on a cryo-handle. Our study firstly assessed the efficiency of cryoprotectant exchange through comparing the osmolalities of fresh and collected solutions during automated vitrification and thawing, and evaluated the cooling and warming rates of the cryo-handle. We also compared mouse oocyte survival, fertilization and embryo development after thawing and ICSI, and the development of re-frozen cleavage embryos between manual operation and automated system. The results showed that the osmolalities of collected samples were within normal range and comparable to fresh solutions. Furthermore, the automated system could obtain the reliable cooling and warming rates. Particularly, there were no significant differences in oocyte survival rates, fertilization rates, and subsequent embryo development and its quality between two procedures. Our findings suggest that AVTS has no impact on osmolalities of vitrification and thawing solutions, ensuring the proper exchange of cryoprotectants. The cryo-handle also shows the ability to achieve reliable cooling and warming rates, which benefits for the cryopreservation and thawing process. Moreover, the results from mouse oocytes and embryos indicate that automated system has effectively maintained the survival and fertilization of frozen oocytes and supported subsequent embryo development. Therefore, the automated vitrification and thawing system will inevitably represent a superior alternative to manual operation.

Biological roles of l-carnitine in oocyte and early embryo development.

Poor oocyte quality is responsible for female infertility. Multiple studies have been carried out to find supplements to enhance oocyte quality and mitigate infertility problems. l-carnitine and its derivatives have diverse roles in developing oocytes and early embryos. This review focuses on the in vitro and in vivo studies that using l-carnitine alone or in combination with other supplements for oocyte quality enhancement. The key roles of l-carnitine in oocyte quality and embryo growth were summarized, and the underlying mechanism was also elucidated. l-carnitine helps in the lipid metabolism process by controlling the transfer of fatty acids to mitochondria for ß-oxidation. l-carnitine modulates glucose metabolism and enhances respiratory chain enzyme activity. Furthermore, it acts as an antioxidant to prevent oxidative damage and inhibit apoptosis, a signal in response to oxidative stress. Results show the potential of l-carnitine as a potential agent in assisted reproductive technology to improve oocyte quality and the subsequent embryonic development.

Follicular dynamics of glycerophospholipid and sphingolipid metabolisms in polycystic ovary syndrome patients.

Polycystic ovary syndrome (PCOS) is a common heterogeneous disease, affecting up to 5-10% women at reproductive age. Although PCOS patients could produce morphologically normal metaphase II oocytes undergoing assisted reproductive techniques (ART), oocyte developmental competence and embryo development have been impaired in following in-vitro fertilization (IVF) steps. Follicular fluid (FF) provides a variety of information in oocyte environment when oocytes grow. In the present work, based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), the metabolic signatures of PCOS FF have been compared with healthy women using untargeted metabolomics approach. Significant abundance differences of a series of glycerolipid, glycerophospholipids, sphingolipids, and carboxylic acids have been discovered. Among them, reduced levels of phosphatidylglycerolphosphate (PGP) and a triglyceride (TG) were highly related to the lower fertilization rate in PCOS; increased abundance of lysoPE and decreased amount of PC were significantly correlated with LH/FSH (ratio of luteinizing hormone to follicle stimulating hormone). Some metabolites, including decreased sphingolipids, glycerophospholipids, and fluctuated fatty acyls, also performed close relationship with other ART and clinical results. We concluded that dysfunctions in the metabolism of glycerolipid, glycerophospholipid, sphingolipid, and glycosphingolipid biosynthesis in PCOS patients' follicles play a non-ignorable role in declining the 2 pronuclei (PN) fertilization rate during IVF procedure.

Ovulation, fertilization and preimplantation embryonic development in raccoon dogs (Nyctereutes procyonoides).

A study involving 32 sexual mature females was conducted to characterize ovulation, fertilization and early embryonic development in vivo in raccoon dogs. Oocytes and embryos were collected from the oviducts and uteri, evaluated by stereomicroscopy. Ovulation occurred 25-32h after a female first accepted mounting, regardless of copulation, when the females were paired with a male in the same cage. Ovulated oocytes were at the primary stage. The number of ovulated eggs in females with or without mating was 9.96±2.65 and 9.00±1.92, respectively. Embryos at 2-4 cell, 8-16 cell, morula, blastocyst, and hatched blastocyst stage were observed at 29-73, 48-100, 98-126, 169-198 and 217-268h after first mating, respectively. Embryos were located in the oviduct prior to 4-cell stage and moved into the uterus after 16-cell stage. Embryos at different stages were often obtained from the same female. During the zygote underwent a series of cleavage divisions, the diameter of the embryo cell mass continuously increased through the 2-cell and 4-cell stage, then started to decrease and was the minimum size at the morula stage. At the blastocyst stage, embryos increased in volume, and finally developed into a hatching blastocyst with a thinner zona pellucida. This is the first full report of preimplantation embryonic development in the raccoon dog, which will facilitate the application of advanced assisted reproductive technology in canine species.

Comparison of different stimulation protocols used in in vitro fertilization: a review.

Infertility is one of the major medical problems in the western world caused by genetic or epigenetic factors, or both, which has led to continuous research and advancements in the field of assisted reproductive technology (ART). Many stimulation protocols are available for controlled ovarian hyperstimulation (COH) in in vitro fertilization (IVF). This review compares the agonist long protocol, antagonist protocol and minimal stimulation protocol. Gonadotropin-releasing hormone (GnRH) antagonist and minimal stimulation protocol has shorter duration of treatment and less gonadotropin use. GnRH agonist long protocol is better in folliculogenesis and pregnancy rate, which is the imperative goal of COH. Despite its costly and lengthy approach, GnRH agonist long protocol has delivered satisfactory results in most women. On the other hand, patients with poor ovarian reserve may have greater advantage when considering minimal stimulation protocol. Evidently, it is crucial to have a larger scale studies with more focused comparisons, which take into account the differences in patients' response criteria and additional confounding variables (age, BMI, previous IVF outcomes etc.), in order to reach to a more definite conclusions.

Assisted reproductive technology in China: compliance and non-compliance.

According to the WHO, infertility and sterility will be the third-most serious disease worldwide in the 21st century, after cancer and cardiovascular diseases. In contrast to developed countries, assisted reproductive technology (ART) were not offered in China until the mid-1980s with the first in vitro fertilization (IVF) infant born in Taiwan in 1985, then Hong Kong in 1986, and mainland China in 1988, respectively. Since those inceptions, the practice of ART in China has evoked a variety of social, cultural, political and one-child policy responses that have resulted in restrictions on the number of IVF cycles performed annually. According to recent survey, an estimate 40-50 million women and 45 million men suffered from infertility, which is estimated that more than ten million Chinese infertile couples require ART treatment. However, it has limited access to ART facilities, many of them may not have a child are whirling to all types of fertility therapies. Exposure to radiation, pesticides and other environmental pollutants, work-related stress and unhealthy lifestyles are believed to contribute to the increasing incidence of infertility in China. The aim of this first report is to provide China nationwide ART data and government policy in compliance and non-compliance, particularly related to family plan policy in China.

Impact of gonadotropins on oocyte maturation, fertilisation and developmental competence in vitro.

The aim of the present study was to evaluate the dose-dependent effects of gonadotropins, either singly (Bravelle (B), Luveris (L), Menupur (M), Repronex (R), Gonal-F (G), Follism (F) and Norvarel (N)) or in combination (Menupur+Bravelle; Repronext+Bravelle; and Bravelle+Norvarel), on rates of oocyte maturation, fertilisation and early embryo development in vitro in an animal model. Bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in TCM-199 with 10% fetal bovine serum supplemented with varying concentrations of gonadotropin (0, 5, 10, 20, 40IU or United States Pharmacopoeia (USP) mL-1) for 24 and 48h according to current IVF clinical stimulation protocols. All gonadotropins enhanced oocyte maturation in vitro in a dose-dependent manner. Individually, Gonal-F (Merck KGaA, Darmstadt, Germany), Follism (Merck Co, Whitehouse Station, NJ, USA) and Repronext (Ferring, Parsippany, NJ, USA) promoted oocyte maturation; in combination, they effectively enhanced COC expansion and increased the maturation competence of MII oocytes. However, high concentrations of gonadotropins may result in maturation arrest. Specific combinations of gonadotropins may change the rate of early embryonic development (8-16-cells) and morula-blastocyst formation. These data provide support for the responsiveness of bovine oocytes to gonadotropins in vitro and the need to consider variations in the relative concentrations and ratio of combinations (FSH/LH or human chorionic gonadotropin) for optimisation of oocyte developmental competence. The results of the present study could be applied to therapeutic clinical stimulation protocols and help improve IVF success rates.

Prevalence of polycystic ovary syndrome in women in China: a large community-based study.

STUDY QUESTION: What is the prevalence of polycystic ovary syndrome (PCOS) in Han Chinese women from different communities? SUMMARY ANSWER: The prevalence of PCOS in Chinese women aged 19-45 years is 5.6%. WHAT IS KNOWN ALREADY: The prevalence of PCOS is reported to range from 5 to 10% but to the best of our knowledge the Han Chinese population has not been studied. STUDY DESIGN, SIZE, DURATION: A large-scale epidemiological study was carried out between October 2007 and September 2011 in 15 924 Han Chinese women of reproductive age (19-45 years) from the 10 provinces and municipalities in China. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 16 886 women from 152 cities and 112 villages were involved in the study. All study participants received a questionnaire and underwent a physical and transvaginal ultrasound examination. Blood samples were collected from a subsample of women (n = 3565) for analysis of metabolic markers and hormones. Based on the Rotterdam PCOS criteria, we assessed hyperandrogenism (H), chronic anovulation (O) and polycystic ovaries (P). Following diagnosis, women with PCOS were assigned to one of four different phenotypes. Finally, the prevalence and related risks of PCOS among Chinese women were estimated based on all the data sources. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 16 886 women were initially involved in the study and 15 924 eligible participants then completed the study; the overall response rate was 94.3% (15 924/16 886). The prevalence of PCOS in the Chinese community population was 5.6% (894/15 924). Blood samples were analyzed from 833 of these women who were assigned to the four PCOS phenotypes as follows: 19% H + O, 37% H + P, 15% O + P and 29% H + O + P. Comparing the 833 women with PCOS to 2732 women without PCOS indicated that PCOS occurs in younger women (P < 0.05) and these women were prone not only to menstrual problems, hyperandrogenism, PCO and infertility but also metabolic syndrome (MS) and insulin resistance (IR). However, there was no significant difference in the rate of hypertension or hyperlipemia between the two groups. Obese patients with PCOS had a higher rate of MS (16 versus 48%), IR (7 versus 28%), hypertension (8 versus 30%) and hyperlipemia (48 versus 73%) compared with non-obese patients (all P < 0.05), respectively. The rates of metabolic complications in patients with PCOS increased with age. LIMITATIONS, REASONS FOR CAUTION: Age and ethnic origin contribute to the differing manifestations of PCOS; therefore, sampling is one of the most important issues in epidemiological research into PCOS. Owing to the mobility of the Chinese population, the survey among resident populations caused a certain deviation in the age distribution. WIDER IMPLICATIONS OF THE FINDINGS: The prevention and treatment of PCOS, particularly in those who are obese, are essential in Chinese women of reproductive age.

Effect of slow freeze versus vitrification on the oocyte: an animal model.

OBJECTIVE: To determine whether there is a deleterious effect on dynamic events in the nucleus and cytoplasm of oocytes by using different cryopreservation protocols in an animal model. DESIGN: Prospective study. SETTING: University hospitals. PATIENT(S): Not applicable. INTERVENTION(S): Immunostaining and confocal laser scanning microscope techniques were used. MAIN OUTCOME MEASURE(S): The spindle and chromosomal configurations, as well as dynamic changes of the cortical granules (CGs) and mitochondria in different cryogroups. RESULT(S): After thawing/warming of bovine oocytes, CGs became more dispersed in the cytoplasm, particularly in the DMSO group. A significant reduction in normal spindle and chromosomal configurations was observed in all three cryogroups, particularly in the propylene glycol (PROH) group, when compared with the fresh group. Global DNA methylation levels were significantly reduced in the slow and DMSO groups, as compared with the fresh group; however, methylation levels were significantly increased in the PROH group. The proportion of severely apoptotic oocytes was dramatically increased in all three cryogroups, compared with the fresh group. CONCLUSION(S): Overall, results demonstrate that using DMSO as the cryoprotectant is better for preserving the cellular and nuclear integrity of the oocyte. The PROH method makes the oocyte more vulnerable to increased DNA methylation, which may be associated with imprinting gene alteration. This study adds to the increasing body of evidence that cryopreservation protocols vary in their impact upon the oocyte.

Comparison of follicular fluid amphiregulin and EGF concentrations in patients undergoing IVF with different stimulation protocols.

Epidermal growth factor (EGF)-like growth factors, such as amphiregulin (AR) and EGF, have emerged as mediators to propagate Luteinizing hormone (LH) stimulus for the oocyte maturation throughout the preovulatory follicle, because cumulus cells and oocytes express few or no LH receptors. This study was to compare AR and EGF concentrations in follicular fluid (FF) among four controlled ovary stimulation (COS) protocols and to investigate the relationship between FF EGF-like growth factors and COS outcomes. Ninety-five patients who underwent in vitro fertilization-embryo transfer (IVF-ET) were treated by four different COS protocols, including gonadotropin-releasing hormone agonist (GnRH-a) long protocol, GnRH-a ultra-long protocol, GnRH-a short protocol, and GnRH antagonist protocol. FF was taken on oocyte retrieval day. FF AR and EGF concentrations were measured and their correlations with COS outcomes were analyzed. FF AR concentration was significantly different from each other among four COS protocol groups (GnRH-a ultra-long protocol group, 186.12 ng/ml; GnRH-a long protocol group, 128.35 ng/ml; GnRH antagonist protocol group, 108.23 ng/ml; GnRH-a short protocol group, 77.13 ng/ml, p < 0.05). FF AR concentrations were higher in GnRH-a ultra-long and long protocol groups, while number of oocytes retrieval, available embryos, and good quality embryos in these two groups were also significantly higher than GnRH-a short protocol group and GnRH antagonist protocol group. FF AR concentration was positively correlated with available embryos, but negatively correlated with serum LH level on hCG day. FF EGF concentration had no relationship with COS parameters. Different COS protocols might have variable effects on AR synthesis. FF AR might be a good indicator to predict the number of oocytes and embryos. FF AR elevation may result in increasing the number of oocyte retrieval and embryo generation, consequently increased cumulative pregnancy rate.

Embryo manipulation and imprinting.

As the use of assisted reproductive technologies (ART) continues to rise worldwide, it remains of the upmost importance to maintain the safety of those techniques used in ART. Many of these practices are unique to this discipline; as such, it becomes difficult to assess the true risks that the potential offspring may be subjected to under this type of treatment. Removal of oocytes from a woman's body during an in vitro fertilization (IVF) cycle offers an increased opportunity for routine cellular processes to go awry. Specifically, epigenetic modifications and imprinting diseases are rare among the general population; however, although their incidence among IVF-conceived children is also rare, their frequency in this population remains elevated compared with universal rates. Recent investigations have directly attributed their occurrences to the use of ART and IVF to achieve a successful pregnancy. This review discusses the major cellular manipulations of a typical IVF cycle to assess the potential risks versus the reported risks. These manipulations include preimplantation genetic diagnosis and screening, intracytoplasmic sperm injection, ooplasmic transfer, embryo culture, in vitro maturation, and cryopreservation. Oocyte and embryo handling is a delicate part of the IVF process that continues to improve. The safety of those potential improvements is also discussed.

Dose-dependent effects of gonadotropin on oocyte developmental competence and apoptosis.

The aim of the present study was to evaluate the effect of gonadotropins (Gn) on oocyte maturation, developmental competence and apoptosis in an animal model. Bovine cumulus-oocyte complexes (COCs) were matured for 24 h in media supplemented with varying concentrations of Bravelle (B), B + Menopur (B+M) or B + Repronex (B + R) (Ferring Pharmaceuticals, Parsiappany, NJ, USA). Then, nuclear maturation, embryo development, and apoptosis in cumulus cells and oocytes were evaluated. Low to moderate Gn concentrations (75-75 00 mIUmL(-1)) effectively improved nuclear maturation and in vitro development. Higher concentrations of Gn (75 000 mIUmL(-1)) did not have any added beneficial effects and nuclear maturation and blastocyst rates in the presence of these concentrations were comparable to control (P>0.05). Most COCs showed slight apoptosis when exposed to 75, 750 and 75 00 mIUmL(-1) Gn; however, when the concentration was increased to 75 000 mIUmL(-1), the proportion of moderately apoptotic COCs increased. In conclusion, extremely high concentrations of Gn have detrimental effects on oocyte nuclear maturation and embryo development and increase apoptosis in cumulus cells, suggesting the importance of judicious use of Gn in assisted reproductive technologies (ART).

Effect of gonadotropins on dynamic events and global deoxyribonucleic acid methylation during in vitro maturation of oocytes: an animal model.

Although the redistributions of mitochondria and cortical granules and global DNA methylation status were not altered in a dose-response manner, high dosages of gonadotropin induced spindle and chromosomal abnormalities. The present study highlights the importance of judicious use of gonadotropins and can be applied to clinical stimulation protocols to reduce the potential risks.

Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common metabolic dysfunction and heterogeneous endocrine disorder in women of reproductive age. Although patients with PCOS are typically characterized by increased numbers of oocytes retrieved during IVF, they are often of poor quality, leading to lower fertilization, cleavage and implantation rates, and a higher miscarriage rate. METHODS: For this review, we searched the database MEDLINE (1950 to January 2010) and Google for all full texts and/or abstract articles published in English with content related to oocyte maturation and embryo developmental competence. RESULTS: The search showed that alteration of many factors may directly or indirectly impair the competence of maturating oocytes through endocrine and local paracrine/autocrine actions, resulting in a lower pregnancy rate in patients with PCOS. The extra-ovarian factors identified included gonadotrophins, hyperandrogenemia and hyperinsulinemia, although intra-ovarian factors included members of the epidermal, fibroblast, insulin-like and neurotrophin families of growth factors, as well as the cytokines. CONCLUSIONS: Any abnormality in the extra- and/or intra-ovarian factors may negatively affect the granulosa cell-oocyte interaction, oocyte maturation and potential embryonic developmental competence, contributing to unsuccessful outcomes for patients with PCOS who are undergoing assisted reproduction.

c-kit and its related genes in spermatogonial differentiation.

Spermatogenesis is the process of production of male gametes from SSCs. The SSCs are the stem cells that differentiate into male gametes in the testis. in the mean time, the Spg are remarkable for their potential multiple trans-differentiations, which make them greatly invaluable for clinical applications. However, the molecular mechanism controlling differentiation of the Spg is still not clear. Among the discovered spermatogenesis-related genes, c-kit seems to be expressed first by the Spgs thus may play a central role in switching on the differentiation process. Expression of Kit and the activation of the Kit/Kitl pathway coincide with the start of differentiation of Spgs. Several genes have been discovered to be related to the Kit/Kitl pathway. in this review, we have summarized the recent discoveries of c-kit and the Kit/Kitl pathway-related genes in the spermatogenic cells during different stages of spermatogenesis.

Dynamic modulation of cytoskeleton during in vitro maturation in human oocytes.

OBJECTIVE: To investigate the role of cytoskeleton in several important dynamic events during in vitro maturation of human oocytes. STUDY DESIGN: Human germinal vesicle stage oocytes were divided randomly into control and study groups. After cultured for 24 hours, chromatin state and position, spindle formation and migration, cortical granules, and mitochondria distribution were evaluated. RESULTS: In colchicine group, spindles did not form. Cortical granules migrated to the cortex but mitochondria maintained the peripheral distribution pattern in most of the oocytes. In cytochalasin B group, the migration of spindle and chromosomes to the cortex was prohibited. Microfilaments disruption influenced cortical granules migration but not redistribution of mitochondria. CONCLUSION: Meiosis progression could not go beyond metaphase I stage when microtubule or microfilament polymerization was prohibited in human oocytes. The migration of cortical granules to the cortex and redistribution of mitochondria to the inner cytoplasm were mediated by microfilaments and microtubules, respectively.

The effect of semen processing on sperm DNA integrity: comparison of two techniques using the novel Toluidine Blue Assay.

OBJECTIVE: The first goal of this study was to determine the effect that semen processing has on sperm DNA integrity. The second goal was to assess which processing technique (modified swim-up versus density gradient centrifugation) results in a superior sample. DNA integrity was measured using a novel Toluidine Blue Assay. STUDY DESIGN: Side-by-side comparison. MATERIALS AND METHODS: Raw semen samples were collected from thirty-two male individuals and scored for routine semen analysis. Prior to discarding the specimens identical aliquots were divided and processed by density gradient centrifugation and a modified swim-up technique. The Toluidine Blue Assay was used to analyze raw and processed samples. RESULTS: Both density gradient centrifugation and the modified swim-up improved DNA quality compared to the unprocessed sample. However, the modified swim-up technique proved superior. CONCLUSIONS: The swim-up technique generates a sperm sample with better DNA integrity. Should DNA integrity correlate with better pregnancy rates in IUI and IVF, respectively, the swim-up may be the sperm processing technique of choice for these procedures.