Unfertilized Oocytes Research Articles

Long-term hypothermic storage of oocytes of the European common frog Rana temporaria at various pressure regimes in gas mixtures based on oxygen, carbon monoxide, and nitrous oxide

In recent years, the challenge of preserving amphibian biodiversity has increasingly been addressed through technologies for the short-term storage of unfertilized spawn at low positive temperatures. Previously the possibility of using a 6.5 atm gaseous mixture of carbon monoxide and oxygen for prolonged hypothermic preservation of unfertilized oocytes for more than 4 days was shown. This study aimed to investigate the viability of oocytes R. temporaria preserved under conditions of hypothermia at 2.5, 3 and 6.5 excess atm pressure in the various gas mixture compositions (CO, N2O, O2) and pure oxygen. The use of pressure up to 3 excess atmospheres was significantly beneficial compared to 6.5 atm at the 7 days storage period. The results indicate that oxygen pressure is a critical factor in maintaining oocyte viability. Admixing CO or N2O to oxygen reduced variability in the results but did not significantly affect the measured indicators (fertilization, hatching) in the experimental groups. The composition CO + O2 (0.5/3.5 ratio, 3 excess atm) reliably extended the shelf life of viable oocytes, indistinguishable from native controls by fertilization and hatching rates, to 4 days. After 7 days, oocytes exhibited fertilization and hatching rates that were 79 % and 48 % compared to native control. Reducing the pressure of the preserving gas mixture to 3 atm, as utilized in this study, simplifies the practical implementation of gas preservation technology for maintaining endangered amphibian species during breeding in laboratory conditions.

O-186 A novel micro-insemination method, assisted sperm fusion insemination, increases the embryo utilization rate compared to conventional intracytoplasmic sperm injection

Abstract Study question Does a novel micro-insemination method, assisted sperm fusion insemination (ASFI), improve fertilization and embryonic development compared to conventional intracytoplasmic sperm injection (C-ICSI)? Summary answer ASFI showed comparable fertilization and blastocyst formation rates to C-ICSI, while achieving significantly higher embryo utilization rates. Furthermore, there were no degenerated oocytes after ASFI. What is known already We have reported that ASFI yielded fertilization and blastocyst rates similar to those obtained with rescue ICSI performed at 6 hours after in vitro fertilization (IVF). In ASFI, fertilization is achieved by pressing a zona pellucida (ZP)-bound sperm onto the oocyte membrane without breaking the oocyte membrane. However, there was a risk of artificial polyspermy because the sperm were pressed against the unfertilized oocytes after IVF. Moreover, it is uncertain whether fertilization occurred with sperm that penetrated into the oocyte via IVF or that were pressed against the oocyte membrane. Clarifying this requires performing ASFI of the oocyte without IVF. Study design, size, duration We prospectively evaluated the effectiveness of ASFI in a sibling oocyte study including 14 subjects from which at least two metaphase II (MII) oocytes and one immature or degenerated oocyte were retrieved. Between December 2022 and October 2023, 119 MII oocytes were obtained in 22 oocyte retrieval cycles and used in the study. Sibling MII oocytes were randomly assigned to a C-ICSI or ASFI group. The present study was approved by the local ethics committee. Participants/materials, setting, methods In the ASFI group, 10,000 motile sperm were incubated with at least one immature or degenerated oocyte for 3 hours in 100 μL of medium per patient. After incubation, the motile sperm bound to the ZP were aspirated directly using an injection pipette. The heads of these ZP-bound sperm were pressed onto the membrane of MII oocytes for 10 seconds using the injection pipette. C-ICSI was conducted using the conventional method. Main results and the role of chance The mean and standard deviation of age of the women and number of MII oocytes per cycle were 39.9 ± 2.2 years and 5.5 ± 1.8, respectively. All sperm collected from the ZP adhered to the MII oocyte membrane after ASFI. Furthermore, time-lapse imaging revealed that all sperm adhering to the MII oocyte membrane were subsequently incorporated into the oocyte. Comparison of the ASFI and C-ICSI groups did not reveal any significant differences in 2 pronuclear embryo rate (ASFI group: 85.4% vs. C-ICSI group: 71.8%), degeneration rate (0% vs. 5.6%), good-quality embryo rate at day 3 (63.4% vs. 52.9%), blastocyst formation rate (67.6% vs. 51.9%), or good-quality blastocyst (Grade 3BB and above by the Gardner criteria) formation rate (29.4% vs. 15.4%). On the contrary, the embryo utilization rate (defined as the total number of embryos transferred and cryopreserved divided by the number of MII oocytes) was significantly higher in the ASFI group compared to the C-ICSI group (47.9% vs. 25.4%, P < 0.05). Limitations, reasons for caution This study was limited by using the ZP for sperm selection. To expand its applicability, a method to identify sperm capable of fusing with an oocyte without using the ZP must be established. Additionally, since our sample size was small, larger studies are required to confirm our findings. Wider implications of the findings ASFI is expected to enhance the survival rate of oocytes and to increase the number of embryos available for implantation, not only compared to rescue ICSI but also C-ICSI. ASFI could aid in understanding fertilization mechanisms by enabling direct observation of oocyte-sperm fusion during the process. Trial registration number UMIN000050184

O-285 Incompatible gametes: implications for infertility diagnosis and treatment

Abstract Sperm face intense selection in the female reproductive tract (FRT), and in humans usually only very few out of up to hundreds of millions of sperm cells can eventually proceed to the vicinity of an unfertilized oocyte. Traditionally, it has been thought that the primary function of female-induced sperm selection is to eliminate poor-quality sperm from reaching the oocyte. However, recent studies have demonstrated that in many species (including humans) FRT also mediate mate choice at the level of the gametes, which frequently bias fertilization towards the male, which is genetically compatible with the female. Furthermore, emerging evidence indicates that sperm phenotypic and genotypic variation within single ejaculates may allow gamete-level mate choice to act also among high-quality (‘healthy’) sperm and bias fertilization towards genetically compatible sperm haplotypes. I argue that both of these cellular-level mate choice processes can have important consequences for deeper understanding of the mechanistic basis of fertilization and infertility.

The effect of exogenous melatonin administration before superovulation on embryo yield in Assaf ewes.

The aim of this study was to determine the effect of exogenous melatonin administration on transferable embryos by increasing total antioxidant status before superovulation in Assaf ewes. Selected ewes were randomly divided into two equal groups: melatonin (n = 9) and control (n = 9). In the melatonin group, a melatonin implant (18 mg melatonin, Regulin®, Ceva, Turkey) was placed under the skin of the ear 7 days prior to insertion of the progesterone-containing sponge. In the control group, a physiological saline solution was injected under the skin of the ear on the same day. The same superovulation protocol was used in both groups. In addition, blood samples for determination of Glutathione peroxidase, superoxide dismutase, total antioxidant status and total oxidant status concentrations were collected on five different days, including the day of melatonin implant placement (Day-7), vaginal sponge insertion (Day 0), vaginal sponge removal (Day 11), mating (Day 12-13) and uterine flushing (Day 19). Embryos were collected by laparotomy on the 7th day after mating. Uterine flushing taken into petri dishes were scanned under a stereomicroscope, and the quality and developmental stages of the embryos were recorded. In the study, total corpus luteum count and total cell count were found to be higher in the control group than in the melatonin group (p < .05). When the results were evaluated in terms of oxidative stress index, a negative correlation was found between the total number of corpus luteum, number of cells obtained, count of transferable embryos and number of Grade 1 embryos on Day 0. There was also a positive correlation oxidative stress index and the number of unfertilized oocytes on Day-7. As a result, exogenous melatonin administration prior to superovulation during the breeding season is thought to have a negative effect on embryo yield and quality. Therefore, the use of exogenous melatonin in MOET studies during the breeding season is recommended to be investigated in new studies.

Coexistence of Endometriosis and Thyroid Autoimmunity in Infertile Women: Impact on in vitro Fertilization and Reproductive Outcomes

Objectives: The objective of the study was to evaluate the prevalence and impact of impaired thyroid-stimulating hormone (TSH) levels on the reproductive outcomes of in vitro fertilization patients diagnosed with endometriosis and compared to controls without endometriosis. Design: This is a retrospective cohort study on prospectively collected data. Setting: The study was conducted at tertiary care university hospital. Participants: Participants were infertile women with histopathological diagnosis of endometriosis. Methods: For 12 months (January 2018 to January 2019), women were deemed suitable and subsequently divided according to serum TSH levels above or below 2.5 mIU/L and compared to patients without endometriosis. Needed sample size was at least 41 patients for each cohort of women. Co-primary outcomes were the live birth rate (LBR), clinical pregnancy rate (CPR), and pregnancy loss rate (PLR). Results: Overall, 226 women (45 with endometriosis and 181 controls without endometriosis) were included. Diagnoses of Hashimoto thyroiditis were significantly more frequent in women with rather than without endometriosis (14/45 [31.1%] vs. 27/181 [14.9%]; p = 0.012). Similarly, in women with endometriosis, Hashimoto diagnosis rates were higher with TSH ≥2.5 mIU/L compared to TSH <2.5 mIU/L (9/15 [60%] vs.5/30 [16.6%]; p = 0.001) so were the Hashimoto diagnosis rates in control group (women without endometriosis) with TSH ≥2.5 mIU/L compared to TSH <2.5 mIU/L (17/48 [35.4%] vs. 10/133 [7.5%], respectively; p = 0.001). Effect size analysis confirmed an increased risk of Hashimoto thyroiditis in women with endometriosis and TSH ≥2.5 mIU/L compared to women with endometriosis and TSH <2.5 mIU/L (risk ratio [RR] 3.60 [95% CI 1.46–8.86]) and in women with endometriosis and TSH ≥2.5 mIU/L compared to non-endometriotic euthyroid patients (RR 7.98 [95% CI 3.86–16.48]). Dysmenorrhea risk was higher in endometriotic euthyroid women compared to euthyroid patients with no endometriosis (RR 1.87 [95% CI 1.21–2.87]). The risk was still increased in euthyroid women with endometriosis relative to dysthyroid women with no endometriosis (RR 1.97 [95% CI 1.11–3.50]). There were no significant differences between the four groups for CPR, LBR, PLR and retrieved oocytes, immature oocytes, degenerated and unfertilized oocytes, cultured blastocysts, embryos and transferred embryos. Limitations: Limitations of the study were retrospective design, limited sample size, and use of different ovarian stimulation protocol. Conclusions: Thyroid autoimmunity seems more common in women with endometriosis and TSH over 2.5 mIU/L. However, there was no significant impact on in vitro fertilization and reproductive outcomes related to the coexistence of endometriosis, Hashimoto disease, and higher TSH levels. Due to limitations of the study, additional evidence is required to validate the abovementioned findings.

A cytological F1 RNAi screen for defects in Drosophila melanogaster female meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1,450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

Diminished NAD+ levels and activation of retrotransposons promote postovulatory aged oocyte (POAO) death

Death is the fate of postovulatory aged or unfertilized oocytes (POAO) in many animals. However, precise molecular mechanisms are yet to be discovered. Here, we demonstrate that increased amounts of reactive oxygen species (ROS), calcium ion (Ca+2) channels, and retrotransposon activity induce apoptosis, which in turn causes POAO death. Notably, suppression of ROS, Ca+2 channels, and retrotransposons delayed POAO death. Further, we found that the histone H4K12 and K16 acetylation increased via downregulation of NAD+ and NAD+ -dependent histone deacetylase SIRT3. Furthermore, adding NMN, sodium pyruvate, or CD38 inhibition delayed the death of postovulatory aged oocytes. Finally, we demonstrate the conservation of retrotransposon-induced DNA damage-dependent POAO death in higher-order vertebrates. Our findings suggest that POAO mortality is caused by cyclic cascade metabolic interactions in which low NAD+ levels increase histone acetylation by inhibiting histone deacetylases, resulting in an increase in retrotransposons, ROS, and Ca+2 channel activity and thus contributing to DNA damage-induced apoptosis.

99 Effects of unfertilized oocytes and blastocysts in the culture environment on cleavage and blastocyst developmental rates of bovine in vitro embryos

99 Effects of unfertilized oocytes and blastocysts in the culture environment on cleavage and blastocyst developmental rates of bovine in vitro embryos

Chromosomal Aberrations As a Biological Phenomenon in Human Embryonic Development.

Frequent chromosomal abnormalities are a distinctive feature of early embryonic development in mammals, especially humans. Aneuploidy is considered as a contributing factor to failed embryo implantation and spontaneous abortions. In the case of chromosomal mosaicism, its effect on the potency of embryos to normally develop has not been sufficiently studied. Although, a significant percentage of chromosomal defects in early human embryos are currently believed to be associated with the features of clinical and laboratory protocols, in this review, we focus on the biological mechanisms associated with chromosomal abnormalities. In particular, we address the main events in oocyte meiosis that affects not only the genetic status of an unfertilized oocyte, but also further embryo viability, and analyze the features of first cleavage divisions and the causes of frequent chromosomal errors in early embryonic development. In addition, we discuss current data on self-correction of the chromosomal status in early embryos.

Administración de lutropina (r-hLH) y su respuesta en la tasa ovulatoria y embriones viables en ovejas Pelibuey

Objective. To evaluate the influence of lutropin alfa (recombinant human LH, r-hLH) administered at 12 and 24 h post-progesterone device removal (CIDR) on the response of hours to the onset of estrus, ovulatory rate, unfertilized oocytes, transferable embryos and plasmatic levels of LH, in a multiple ovulation protocol based on FSHp in Pelibuey sheep. Materials and methods. Eighteen sheep of Pelibuey sheep were used, which were randomly distributed into three treatments (n=6): T1 (200 mg FSHp-Folltropin®); T2 (200 mg pFSH + 75 IU r-hLH at 12 h post-CIDR withdrawal) and T3 (200 mg pFSH + 75 IU r-hLH at 24 h post-CIDR withdrawal). Results. Hours from start to estrus and ovulatory rate (OR) no significant differences were found. In unfertilized oocytes (UO) it was higher (p&lt;0.05) in T2 (5.0 ± 2.3) compared to T1 (0.8 ± 0.6) and T3 (1.3 ± 0.7). The number of Quality 1 Embryos (Q1E) was higher (p&lt;0.05) in T3 (3.1 ± 1.1) compared to T1 (1.1 ± 0.3) and T2 (0.6 ± 0.5) and for Transferable Embryos (TE) it was higher (p&lt; 0.05) for T3 (6.6 ± 0.9) in relation to T2 (2.0 ± 1) and T1 (4.0 ± 0.6). Conclusions. The application of r-hLH 24 h after removing the CIDR increases plasma levels of LH, in effect, the number of unfertilized oocytes is reduced, and the number and quality of transferable embryos in Pelibuey sheep are improved.

K252a inhibition of sperm viability for efficient crossbreeding of hermaphroditic bivalves

Bivalve mollusks are economically important shellfish, representing 56.3% of aquaculture production. Crossbreeding is a popular approach for the genetic improvement of aquaculture species, including bivalves. However, it is difficult for simultaneous hermaphrodites due to the difficulty in collecting unfertilized oocytes. Male infertility or subfertility could be a solution. Considering that protein phosphorylation is critical in spermatogenesis, we investigated the effects of the protein kinase inhibitor K252a on germ cell development, gamete quality and embryonic/larval development of the simultaneously hermaphroditic scallop Argopecten irradians and evaluated its feasibility in crossbreeding. The results showed that 20% K252a injection at the gonad growth stage led to significant decreases in sperm number and progressive motility rate. Abnormal axoneme arrangement in flagella may have caused reduced sperm motility. Normal oocytes fertilized with K252a-treated sperm showed significant decreases in fertilization rate (38.24%), cleavage rate (83.42%) and D-stage survival rate (15.93%), and K252a-treated oocytes did not show obvious defects in early development. Interspecific hybridization using K252a-treated A. irradians fertilized with Argopecten purpuratus sperm resulted in a 3.19-fold increase in the hybrid ratio (67%) over that of the control group (16%). Our study indicated that K252a influenced sperm development without affecting oocytes in A. irradians, indicating that K252a treatment may facilitate the mass production of crossbreeds and hybrids for simultaneous hermaphrodites. The approach reported here will greatly assist in genetic improvement programs for mollusk hermaphrodites.

Prolonged hypothermic storage of oocytes of the European common frog Rana temporaria in a gas mixture of oxygen and carbon monoxide.

The maximum hypothermic storage time of amphibian oocytes is several hours, which is due to the peculiarities of the structure of the cell envelope. The authors of this paper have already demonstrated the possibility of increasing the storage period of unfertilized oocytes of the common frog (Rana temporaria) up to 5-7 days. The aim of the current study was to determine the possibility of using a 6.5 atm gaseous mixture of carbon monoxide and oxygen, for prolonged hypothermic preservation of unfertilized oocytes for 4 to 12 days. After four days, oocytes stored under CO+O2 conditions exhibited fertilization and hatching rates that were 1.6 and 2.2-fold higher than control, respectively. While no oocytes in the control group survived to day twelve, oocytes held under CO +O2 gas exhibited a 39±14% (38 out of 99 oocytes in total) fertilization rate, however only 1±2% (1/99) of those hatched. This approach is promising for the storage of genetic material from female amphibians, particularly in respect to managing and restoring endangered species, but may also be applicable to oocytes of other classes of vertebrates.

P-116 How does the short insemination method affect clinical outcomes and laboratory management?

Abstract Study question Does the short insemination method using a time-lapse monitoring system improve clinical outcomes and laboratory management? Summary answer The method increased the normal fertilization rate of Conventional IVF and also increased the 3PN rate. The working environment of the laboratory staff was improved. What is known already Short insemination using a time-lapse monitoring system has been reported to reduce fertilization confirmation failures due to loss of pronuclei, but many reports indicate that the method was implemented to perform so-called 'rescue ICSI' for unfertilized oocytes on the day of egg retrieval and there are not many reports of clinical outcomes in conventional IVF without rescue ICSI. There are also few reports on the working environment and working hours in the laboratory. Study design, size, duration This was a retrospective study of 3,714 oocytes from 715 cycles in 595 patients who underwent Conventional IVF with egg retrieval in 2016-2022. Long insemination (1,577 oocytes) was performed in cycles up to May 2019 and short insemination (2,137 oocytes) in later cycles. In IVF, 40,000 motile sperm were placed in 1 ml of culture medium per oocyte. Participants/materials, setting, methods Oocytes of the long insemination group (group L) were inseminated 5 hours after egg retrieval and denuded 19 hours later to observe their pronuclei. Oocytes from the short insemination group (group S) were inseminated 2 hours after egg retrieval, denuded 4 hours later, and cultured in EmbryoScope (Vitrolife, Sweden) to observe the pronuclei. Since the working hours set at our clinic are 8:00 a.m.-5:00 p.m., the time schedule for both groups followed this schedule. Main results and the role of chance The 2PN rate was 60.9% in group L and 64.7% in group S. The 3PN rates were 7.5% and 10.2% respectively, both rates being significantly higher in group S (P &amp;lt; 0.05). The non-fertilization rates were significantly higher in group L (25.3% and 21.7% respectively); the 1PN rate was not significantly different (3.6% vs. 3.4%). The rate of missed pronuclei was 2.8% in group L and 0% in group S. Early embryo transfer pregnancy rate (25.4% vs 27.8%), good blastocyst development rate (52.2% vs 63.6%) and blastocyst transfer pregnancy rate (41.1% vs 31.7%) in groups L and S respectively. The good blastocyst incidence was significantly higher in group S. In the case of the Conventional IVF cycle in which egg retrieval was performed at 8:00 a.m., the embryologists in group L were to observe the pronuclei at 8:00 a.m. the next day, but in reality the embryologists had to come to work earlier because of denudation, and even then pronuclear loss occurred. In contrast, the group S did not require an earlier attendance because denudation was not performed the next morning, and the time-lapse images did not fail to confirm the pronuclei at all. Limitations, reasons for caution Since the short insemination method was introduced at our clinic in June 2019, this study compared clinical outcomes before and after that date. Wider implications of the findings It has been reported in the past that early insemination time increases both 2PN and 3PN rates. The short insemination method in this study also used a faster insemination time, which may have led to similar insemination results. Trial registration number not applicable

P-247 Total Fertilization Failure after conventional IVF: it is not the end of the story

Abstract Study question Could Rescue IntraCytoplasmatic Sperm Injection (R-ICSI) coupled with frozen transfers be an effective treatment for Total Fertilization Failure (TFF) after conventional InVitro Fertilization (c-IVF) cycles? Summary answer Applying R-ICSI after TFF of c-IVF, a blastulation rate of 28% could be obtained, allowing the cycle rescue in a consistent proportion of couples. What is known already Conventional IVF is to date the most indicated technique in infertile couples in the absence of male factor. However, the incidence of TFF following c-IVF, in presence of normozoospermic samples, ranges from 5 to 20%. TFF leaves experts with limited alternatives, which include cancelling the cycle with subsequent indication to proceed with ICSI in the next cycle or attempting R-ICSI in the current cycle. R-ICSI coupled with frozen embryo transfer may overcome most of the technical and biological issues associated with fresh transfer after R-ICSI, possibly representing an efficient procedure for couples experiencing TFF following c-IVF. Study design, size, duration Our retrospective study included 33 couples who underwent assisted reproduction techniques (ART) at the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico of Milan from October 2021 to December 2022 and that experienced TFF after c-IVF, regardless the number of oocytes retrieved. Overall, TFF was reported in 7% of c-IVF cycles (33/439). An indication for male factor infertility was an exclusion criterium. World Health Organization (WHO) reference values for human semen characteristics have been adopted. Participants/materials, setting, methods Fertilization failure was considered in terms of absence of the two pronuclei and the second polar body. Out of 158 unfertilized oocytes, 128 MII oocytes were microinjected after c-IVF in the same cycle. R-ICSI consists of performing ICSI 22-24 hours after insemination with an unsuccessful c-IVF, using the same semen sample maintained at room temperature overnight. Fertilized oocytes after R-ICSI were cultured to the blastocyst stage according to our standard protocol. Main results and the role of chance The fertilization rate was 42% (95%CI: 34-51%). Fertilization failure was still observed in 9 patients (27%, 95%CI: 13-46%) after R-ICSI, with a median value of 2 [2-5] oocytes available for insemination. In the 24 patients who obtained at least one zygote, the blastulation rate was 28% (95%CI: 16-42%). A total of 15 blastocysts were cryopreserved in 10 patients on day 5 or 6 after R-ICSI. Of these 10 patients, 6 have undergone frozen/thawed single embryo transfer, and the success rate (ongoing pregnancy) was 50% (95%CI: 12-88%). Limitations, reasons for caution One of the limitations is the small sample size, since this is an emergency procedure. However, the study is still ongoing. Women were not subjected to inclusion criteria, thus fertilization failure could be attributed to several factors different from semen quality. Wider implications of the findings This study turns out to be important in light of the interesting implantation rate obtained. With the advantage of reducing both costs and psychological burden of the couple, the technique can be proposed as a rescue strategy in cases of TFF allowing to confidently increase the number of c-IVF procedure. Trial registration number not applicable

In silico identification and characterization of the SNPs in the human ASTL gene and their probable role in female infertility.

Ovastacin (ASTL), a zinc metalloprotease, is released from a fertilized egg during exocytosis of cortical granules which occurs minutes after the sperm and egg fuse. ASTL cleaves ZP2, one of the four primary glycoproteins of human zona pellucida, and this cleavage prevents polyspermy, causes zona pellucida hardening, and also protects the pre-implantation embryo. Any perturbation in the activity of ASTL can thus disturb this process and may lead to infertility without changing the gross morphology of the oocyte. A small amount of ASTL is also released by unfertilized oocytes but its catalytic activity is absent as it is bound by its inhibitor, Fetuin-B (FETUB). Pre-mature release of ASTL when FETUB is absent also causes infertility. To identify and understand the structural and functional effects of deleterious SNPs of ASTL on its interaction with ZP2 and FETUB and hence on fertility, a total of 4,748 SNPs from the dbSNP database were evaluated using a variety of in silico tools. All of the 40 shortlisted nsSNPs were present in the catalytic domain of the protein. Comparison of the wild type with mutants using MutPred2 suggests an alteration in the catalytic activity/zinc binding site in many SNPs. Docking studies show the involvement of hydrophobic interactions and H bonding between ASTL and ZP2 and also between ASTL and FETUB. Four positions in ASTL involved in the hydrophobic interactions (P105 and D200 between ASTL and ZP2; D198 and L278 between ASTL and FETUB) and 5 in H bonding (E75 and R159 between ASTL and ZP2; and K93, R159, and C281 between ASTL and FETUB) have SNP's associated with them validating their importance. Interestingly, a cluster of multiple SNPs was found in the motif 198DRD200, which is also a well-conserved region among several species. Statistical Coupling Analysis (SCA) suggested that the deleterious SNPs were present in the functionally important amino acid positions of ASTL and are evolutionarily coupled. Thus, these results attempt to identify the regions in ASTL, mutations in which can affect its binding with ZP2 or FETUB and cause female infertility.

Fertility and fertility preservation in women

Advances in the treatment of cancer and in reproductive medicine make it possible for many patients to start their family planning even after cytotoxic therapy. Depending on the age of the patient, the planned oncological therapy and its urgency, various methods can be used to preserve the fertility of affected women. Presentation of facts about fertility, as well as information about fertility-preserving methods for women, so that they can be discussed with and offered to patients. Presentation and discussion of basic research, clinical data, and expert recommendations on fertility and fertility preservation. Well-established fertility-protective techniques now exist for women that offer arealistic chance of subsequent pregnancy. These include transposition of the gonads prior to radiotherapy, gonadal protection with gonadotropin-releasing hormone (GnRH) analogues and cryopreservation of fertilized and unfertilized oocytes, as well as cryopreservation of ovarian tissue. Fertility-protective techniques are an integral part of oncological treatments for prepubertal girls and patients of reproductive age. The various measures must be discussed individually with the patient as part of amultimodal concept. Prompt and timely collaboration with aspecialized center is essential.

Biases in quantitative genetic parameters estimations caused by self-fertilization in the Caribbean pectinid Argopecten nucleus (Mollusca: Bivalvia)

Inbreeding can cause an important bias in the estimates of quantitative genetic parameters, which in turn can affect the estimated potential for a selection response in species that are simultaneous hermaphrodite with partial self-fertilization. In the present study, the biases caused by self-fertilization on heritability (h2) estimates for productive traits were assessed at different ages in the tropical hermaphrodite pectinid Argopecten nucleus. Self-fertilization rates were estimated in both oocytes and adults, as the percentage of zygotes formed from unfertilized oocytes and by paternity analysis using microsatellite genetic markers, respectively. Fifty sexually mature individuals were individually spawned and genotyped using microsatellite markers. The sperm of one individual used as a male was used to fertilize the oocytes of 4 other individuals, generating 10 half-sibling families (HS) and 40 full-siblings families (FS). At the age of 3.5 months, the total weight, and the length, height, and width of the shell of 60 animals per FS family were individually measured. At the age of 9 months, the same measurements were repeated in the same individuals, which were then euthanized to weigh the soft tissues. In addition, the fecundity was estimated in each individual (number of oocytes produced), and a paternity analysis was performed. No significant differences were found in the average inbreeding rates estimated in oocytes and adults. However, no significant correlation was observed in the estimates of family self-fertilization rates when using both methods. The individuals derived from crossbreeding showed higher average values for productive traits than those produced by selfing, although interactions between the genotype and the depth of culture were observed in both groups. The h2 estimates using statistical models that consider self-fertilization in adults (models II and III) were lower than those observed when self-fertilization rates were estimated in oocytes, or not included in the model. However, the genetic correlations between the traits were similar using both methods for estimating self-fertilization rates. The present results suggest that self-fertilization results in inbreeding depression and induces an overestimation of the h2 for productive traits, but it does not affect estimates of genetic correlations or genotype by environment interactions.

Effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen

Superovulation is a drug-based method used in cattle to stimulate the ovarian folliculogenesis and the number of oocytes and transferable embryos. The present study aimed to test the effects of recombinant FSH (bscrFSH) and pituitary FSH (FSH-p) on ovarian response and in vivo embryo production in superovulated dairy heifers inseminated with unsorted and sex-sorted semen. Forty healthy Holstein heifers subjected to a superovulation (SOV) protocol by using FSH-p or bscrFSH were divided randomly into four groups: a) FSH-p inseminated with unsorted semen (USP; n = 10), b) FSH-p inseminated with sex-sorted semen (SSP; n = 10), c) bscrFSH inseminated with unsorted semen (USR; n = 10), and d) bscrFSH inseminated with sex-sorted semen (SSR; n = 10). Ultrasonography was carried out on Day 8 (estrus) and Day 15 (embryo collection) to evaluate the ovarian structures [follicles (FL), corpora lutea (CL), and non-ovulated follicles (NOFL)]. Embryonic-derived parameters were scored on Day 15 [total structures collected (TS), unfertilised oocytes (UFOs), total embryos (TEs), transferable embryos (TFEs), freezable embryos (FEs), and degenerated embryos (DEs)]. No differences were observed regarding ovarian structures (FL and NOFL) irrespective of SOV protocol or group assessed (P > 0.05). CL increased in bscrFSH-derived SOV protocol (P < 0.05). On Day 15, the embryonic-derived parameters TEs, TFEs, and FEs decreased in SSP/SSR compared to USP/USR (P < 0.05). Differences were observed regarding UFOs, with a greater number in SSP and SSR (P = 0.01). In conclusion, the bscrFSH-derived SOV protocol showed improved results compared to FSH-p-derived SOV protocol regarding ovarian (CL) and embryo-derived (TFE) parameters irrespective of the type of semen used.

Potential role for human menopausal gonadotropin in ovine superovulatory regimens during the breeding season.

Human menopausal gonadotrophin (hMG) has been reported to produce a comparable superovulatory response to that of follicle stimulating hormone (FSH). Furthermore, hMG has a long half-life as compared with FSH. The present study was designed to compare hMG administered once daily and FSH administered twice daily over a 4 - day period on superovulatory response of Suffolk ewes. During the mid-luteal phase, twenty-four Suffolk donor ewes received intravaginal sponges at day 0 for 12 days. The superovulatory regimens in the Control group (n=12) and the Treatment group (n=12) consisted of eight injections of FSH given at twice daily and four injections of hMG given at once daily, respectively. At day 13, the donor ewes were subjected to laparoscopic insemination. Embryos were recovered, classified, and transferred to recipient ewes at day 19. Pregnancy status was determined by ultrasound examination 40 days after transfer. Lambing rate was calculated after all the ewes had delivered. No significant differences were observed between the two groups in terms of the structures recovered, transferable embryos, degenerated embryos, unfertilized oocytes, pregnancy rate and lambing rate. The results showed that once daily injection of hMG can produce a comparable superovulatory response and embryo transfer outcomes to those obtained by twice daily injection of FSH over a 4 - day period. It is feasible that hMG is used to replace FSH and reduce the number of injection treatments in ovine superovulatory regimens.

Characteristics of oocytes that failed to fertilize after intracytoplasmic sperm injection.

We assessed the morphologies of meiotic spindles in oocytes that failed to fertilize following intracytoplasmic sperm injection (ICSI) and identified factors contributing to failed fertilization. A total of 225 unfertilized oocytes were collected after ICSI. Oocytes were fixed and stained for tubulin and chromosomes. Meiotic spindle morphologies, chromosome alignment, and sperm nuclear decondensation were assessed to identify contributing factors to fertilization failure. We identified relationships between several factors and both abnormal spindle morphologies and sperm nuclear decondensation in oocytes that failed to fertilize. Three causes for unfertilized oocytes after ICSI were identified: (I) the absence of a sperm nucleus in the ooplasm; (II) failed oocyte activation; and (III) defects in pronucleus formation or migration. The rate of disarranged polar spindles in oocytes collected from women older than 35 years (73.3%; 33/45 oocytes) was significantly higher than that of those collected from women 35 years and younger (50.4%; 68/135 oocytes; odds ratio [OR]: 2.71, 95% confidence interval [CI]: 1.29-5.69, p = 0.009). The proportion of unfertilized oocytes with abnormal spindles and chromosome misalignment was significantly higher in oocytes collected from women older than 35 years than those from women 35 years and younger (62.2% vs. 41.5%, p = 0.016). The proportion of partially decondensed chromatin in the abnormal sperm morphology group was significantly higher than in the normal sperm morphology group (66.7% versus 52.9%, OR: 1.78, 95% CI: 1.01-3.11, p = 0.044). The main contributor to the failure of oocytes to fertilize after ICSI is failed oocyte activation. The ICSI technique used, the maternal age, and sperm morphology are also contributing factors in fertilization failure after ICSI.