Genetic screening and analysis of TUBB8 variants in females seeking ART

Research question: More than 100 variants have been identified in the TUBB8 gene, which account for approximately 30% of infertile women with oocyte maturation defects. But what is the correlation between the highly phenotypic diversity and genetic variability? Are there other variants in TUBB8 related to female infertility?Design: TUBB8 resequencing was performed in 80 female subjects who were experiencing infertility and were seeking treatment with assisted reproductive technologies (ART), or had ever experienced ART failure due to oocyte maturation defects. All variants were evaluated with pedigree analysis, population frequency, in-silico analysis and molecular modelling. The effects of the variants on oocytes/arrested embryos were assessed by morphological observations, immunostaining, embryo biopsies and chromosome euploidy analysis.Results: Nine missense variants and two frameshift variants from an additional 15 families were identified, including four novel variants and seven previously reported recurrent variants. These TUBB8 variants were related to highly variable phenotypes, including abnormalities in oocyte maturation or morphology, fertilization failure, embryonic development abnormalities and implantation failure. Also further clarified were the incomplete penetrance of heterozygous p.E108K, the likely benign significance of heterozygous p.A313V and the clinical effect of a novel variant of p.R380C.Conclusions: This study significantly expands the variant spectrum of the TUBB8 gene and, together with the available findings on TUBB8 variants and female infertility, will potentially facilitate the genetic counselling of infertile women in future.     

Mutation analysis of the TUBB8 gene in primary infertile women with arrest in oocyte maturation

Tubulin beta eight class VIII (TUBB8) is a subtype of β-tubulin that only exists in primates. Mutations in the TUBB8 gene have been proven to cause oocyte maturation arrest. The aim of this study was to identify the new types of mutations in TUBB8. Six women (families) with oocyte maturation arrest and 100 healthy controls were recruited. The sequence of the TUBB8 gene was amplified and analyzed by Sanger sequencing, which revealed a de novo heterozygous variant c.292G?>?A (p.G98R) of TUBB8 in one affected individual. This TUBB8 variant was absent in the 100 fertile females and was predicted to be highly damaging to the function of the TUBB8 protein by SIFT and PolyPhen-2. This novel variant extends the spectrum of TUBB8 mutations and the presence of a TUBB8 mutation is being considered to be indicative of a poor prognosis for the success of assisted reproductive treatment.     

Reduced In Vitro Fertilization of Human Oocytes Correlates with Raised Circulating FSH Levels During Ovarian Stimulation in Normogonadotropic Women Downregulated with GnRH-Analogues

Purpose: The possible effects of circulating FSH levels as used during IVF treatment on oocyte maturation and subsequent preembryo development were evaluated. Methods: Serum levels of FSH and LH on days 1 and 8 of ovarian stimulation and on the day of oocyte retrieval (OR) were correlated with subsequent preembryo development in vitro. After pituitary downregulation, 244 normogonadotropic women followed a fixed protocol for the first 7 days of stimulation. Results: The average FSH concentration on day 8 of stimulation was 11.5 IU/L and exceeded the expected midcycle surge of FSH by more than 25%. In contrast, levels of LH were below an average of 2 IU/L throughout the stimulation period. The concentration of FSH on day 8 and on the day of OR showed a significant inverse correlation with cleavage rate, whereas levels of LH, age, and body mass index showed no such correlation. Conclusions: Supraphysiologic levels of FSH seems to affect oocyte maturation negatively. Premature resumption of meiosis, leading to retrieval of postmature oocytes with a reduced developmental potential, is suggested as the underlying mechanism.     

Live birth rate from euploid blastocysts is not associated with infertility etiology or oocyte source following frozen-thawed embryo transfer (FET): analysis of 4148 cycles reported to SART CORS

PurposeTo investigate whether live birth rates from euploid blastocyst frozen-thawed embryo transfer (FET) cycles are associated with infertility diagnosis or oocyte source.DesignRetrospective analysis of FET cycles reported to SART CORS in 2014.MethodsData from fresh IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A), linked to the first FET cycles, were collected from the 2014 SART CORS database for autologous and donor oocyte cycles. Inclusion criteria were patients undergoing FET with euploid embryos (n = 4148). Demographic data including age, BMI, prior fertility, and etiology of infertility were collected from the retrieval cycle and analyzed. Patients with uterine anomalies, preimplantation genetic testing-mutation (PGT-M) for genetic diseases, gender selection, HLA determination, or systemic and immunologic disorders were excluded. The primary outcome measure was live birth (LB) rate. Potential confounders such as age, prior fertility, and maximum baseline FSH values were analyzed with regression models as indicated.ResultsThough age, maximum baseline FSH, and infertility diagnosis were significantly different, LB was similar between patients undergoing autologous or donor oocyte FET cycles. Etiology of infertility was not significantly associated with LB in autologous cycles (p = 0.95). Potential confounders such as maternal age, prior fertility, and maximum baseline FSH were not associated with outcomes; however, maternal BMI was inversely related to LB in autologous cycles, with an odds ratio of 0.97 (95% CI: 0.96–0.98 (rho = − 0.08, p < 0.01)).ConclusionsAfter controlling for confounding variables, a euploid embryo derived from a donor or autologous oocyte results in similar LB in women with different infertility diagnoses.     

The Potential Use of Maturation In Vitro of Human Oocytes in Low Responder Patients

Purpose: To assess whether maturation in vitro of humanoocytes (MIVHO) could be an alternative treatment in lowresponders to ovarian stimulation for in vitro fertilization(IVF). Methods: Prospective case=ncontrol study. Spontaneouslyovulatory women who volunteered were included in ourprogram of MIVHO at the Instituto Valenciano deInfertilidad. Rates of oocyte retrieval, in vitro maturation,fertilization, and development up to the blastocyst stage werestudied. Results: A significantly increased rate of oocyte retrievalwas found when the pickup was performed before follicularselection. No differences were found when MIVHO was usedin a low responder patient with an ovarian content of earlyantral follicles > 5 as compared to normal responders. Conclusions: MIVHO could be a successful choice in lowresponder patients with an acceptable number of early antralfollicles. Oocyte retrieval should be performed beforefollicular selection in order to obtain more oocytes.     

Genetic factors as potential molecular markers of human oocyte and embryo quality

Successful human reproduction requires gamete maturation, fertilization, and early embryonic development. Human oocyte maturation includes nuclear and cytoplasmic maturation, and abnormalities in the process will lead to infertility and recurrent failure of IVF/ICSI attempts. In addition, the quality of oocytes/embryos in the clinic can only be determined by morphological markers, and there is currently a lack of molecular markers for determining oocyte quality. As the number of patients undergoing IVF/ICSI has increased, many patients have been identified with recurrent IVF/ICSI failure. However, the genetic basis behind this phenotype remains largely unknown. In recent years, a few mutant genes have been identified by us and others, which provide potential molecular markers for determining the quality of oocytes/embryos. In this review, we outline the genetic determinants of abnormalities in the processes of oocyte maturation, fertilization, and early embryonic development. Currently, 16 genes (PATL2, TUBB8, TRIP13, ZP1, ZP2, ZP3, PANX1, TLE6, WEE2, CDC20, BTG4, PADI6, NLRP2, NLRP5, KHDC3L, and REC114) have been reported to be the causes of oocyte maturation arrest, fertilization failure, embryonic arrest, and preimplantation embryonic lethality. These abnormalities mainly have Mendelian inheritance patterns, including both dominant inheritance and recessive inheritance, although in some cases de novo mutations have also appeared. In this review, we will introduce the effects of each gene in the specific processes of human early reproduction and will summarize all known variants in these genes and their corresponding phenotypes. Variants in some genes have specific effects on certain steps in the early human reproductive processes, while other variants result in a spectrum of phenotypes. These variants and genetic markers will lay the foundation for individualized genetic counseling and potential treatments for patients and will be the target for precision treatments in reproductive medicine.     

Intracytoplasmic Sperm Injection After Follicle Stimulation with Highly Purified Human Follicle-Stimulating Hormone Compared with Human Menopausal Gonadotropin

Purpose: Our purpose was to compare oocyte nuclear maturation and embryo quality after pituitary down-regulation and ovarian stimulation with highly purified follicle-stimulating hormone (FSH) or human menopausal gonadotropin (HMG). Methods: Fifty-five patients 37 years of age or younger who were undergoing in vitro fertilization (IVF)–intracytoplasmic sperm injection (ICSI) were evaluated retrospectively. In all cases, male factor was the only indication for treatment, with no female-related factors identified. Following pituitary down-regulation, patients were stimulated with hMG (n = 20) or highly purified FSH (n = 35). Main outcome measures included ovarian response to stimulation, oocyte maturity, and ICSI fertilization results. Secondary outcome measures included pregnancy rates and outcome. Results: The ovarian response to stimulation was similar for the two groups, as were the percentage of metaphase II oocytes, fertilization and cleavage rates, and number and quality of transferred and cryopreserved embryos. Cycle outcome was comparable. Conclusions: In normogonadotropic subjects, monocomponent therapy with highly purified FSH is as effective as hMG in stimulating ovarian follicular development, synchronization of oocyte maturation, and IVF-ICSI outcome. Our findings support the conclusion that the luteinizing hormone component in the stimulation protocol is unnecessary.     

Ovarian stimulation for oocyte cryopreservation for prevention of age-related fertility loss: one in five is a low responder

Abstract

Oocyte cryopreservation for age-related fertility loss is gaining interest considering the tendency to postpone motherhood in many societies. Little is currently known about the actual efficiency of this approach. We aimed to explore ovarian response of presumably fertile women undergoing in vitro fertilization for this indication. A total of 105 women underwent 151 stimulation cycles at mean age 37.7?±?2.4. None had known infertility. Mean daily starting FSH dose was 371?±?110 (225–600). Mean number of mature oocytes cryopreserved at the first completed cycle was 9.7?±?7.5 (0–43). However, 21% of started cycles were either cancelled before egg retrieval or resulted in 0–3 mature oocytes retrieved. Therefore, women considering oocyte cryopreservation for prevention of age-related fertility decline should be encouraged to perform this procedure at younger age than, preferably before 35.     

Oocyte Cryopreservation for Fertility Preservation in Postpubertal Female Children at Risk for Premature Ovarian Failure Due to Accelerated Follicle Loss in Turner Syndrome or Cancer Treatments

ObjectiveTo preliminarily study the feasibility of oocyte cryopreservation in postpubertal girls aged between 13 and 15 years who were at risk for premature ovarian failure due to the accelerated follicle loss associated with Turner syndrome or cancer treatments.DesignRetrospective cohort and review of literature.SettingAcademic fertility preservation unit.ParticipantsThree girls diagnosed with Turner syndrome, 1 girl diagnosed with germ-cell tumor. and 1 girl diagnosed with lymphoblastic leukemia.InterventionsAssessment of ovarian reserve, ovarian stimulation, oocyte retrieval, in vitro maturation, and mature oocyte cryopreservation.Main Outcome MeasureResponse to ovarian stimulation, number of mature oocytes cryopreserved and complications, if any.ResultsMean anti-müllerian hormone, baseline follical stimulating hormone, estradiol, and antral follicle counts were 1.30 ± 0.39, 6.08 ± 2.63, 41.39 ± 24.68, 8.0 ± 3.2; respectively. In Turner girls the ovarian reserve assessment indicated already diminished ovarian reserve. Ovarian stimulation and oocyte cryopreservation was successfully performed in all female children referred for fertility preservation. A range of 4-11 mature oocytes (mean 8.1 ± 3.4) was cryopreserved without any complications. All girls tolerated the procedure well.ConclusionsOocyte cryopreservation is a feasible technique in selected female children at risk for premature ovarian failure. Further studies would be beneficial to test the success of oocyte cryopreservation in young girls.     

The different impact of stimulation duration on oocyte maturation and pregnancy outcome in fresh cycles with GnRH antagonist protocol in poor responders and normal responders

ObjectiveTo study the impact of stimulation duration on intracytoplasmic sperm injection (ICSI) - embryo transfer (ET) outcome in poor and normal responders during controlled ovarian stimulation using gonadotropin-releasing hormone (GnRH) antagonist protocol.Materials and methodsThis is a retrospective cohort study. There were 1481 women undergoing ICSI-ET cycles. Women with ovum pick-up number ≤3 were defined as poor responders (n = 235), and those with a number ≥4 were normal responders (n = 1246).ResultsThe mean stimulation duration was shorter in poor responders with pregnancy group as compared with normal responders with pregnancy group (7.8 ± 2.2 vs. 9.2 ± 1.6 days, p < 0.01). Poor responders with a shortest stimulation duration (≤6 days) appeared a higher live birth rate (≤6 days: 33.3%, 7–8 days: 20.0%, 9–10 days: 15.9%, and ≥11 days: 11.1%, p = 0.18). Normal responders with a shortest stimulation duration (≤6 days) appeared a lowest live birth rate (≤6 days: 28.6%, 7–8 days: 35.8%, 9–10 days: 33.6%, and ≥11 days: 29.3%, p = 0.61). Oocyte maturation rate was significantly lower at stimulation durations ≤6 days group (≤6 days: 67%, 7–8 days: 80%, 9–10 days: 85%, and ≥11 days: 87%, p = 0.02) in normal responders.ConclusionIn ICSI-ET cycles, stimulation duration appears to have different impact on oocyte maturation, clinical pregnancy rates and live birth rates in both poor and normal responders.     

Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa

Purpose: In vitro maturation (IVM) of immature human oocytes is an attractive option for the treatment of infertility. Similarly, intracytoplasmic sperm injection (ICSI) followed by testicular fine needle aspiration (TEFNA) is an important treatment for primarily male-factor infertility. This report highlights the combination of these two advanced assisted reproduction techniques, namely IVM and fertilization with TEFNA-retrieved spermatozoa by ICSI to overcome both of male and female infertility problems.Methods: Before immature oocyte retrieval (IOR), gonadotropin stimulation was given for 3 or 5 days. Following IVM, and mature oocytes were inseminated by ICSI followed by TEFNA.Results: Four couples with five completed treatment cycles were performed, and total of 36 immature oocytes were retrieved. Following 36 to 48 h of culture, 32 (88.89%, 32/36) oocytes became mature. The mature oocytes were inseminated with TEFNA-retrieved sperm, and 18 (56.25%, 18/32) oocytes were fertilized normally following ICSI. Eleven embryos were transferred in five cycles and two pregnancies and two singleton births were achieved in two patients.Conclusions: This result demonstrates that the successful pregnancies and live births can be established from embryos produced from {in vitro} matured oocytes that fertilized with testicular sperm.     

The comprehensive variant and phenotypic spectrum of TUBB8 in female infertility

Journal of Assisted Reproduction and Genetics - TUBB8 is a gene that is frequently analysed in the genetic diagnosis of female infertility; 102 variants of this gene have been identified. However,...     

Is there an optimal timing interval between hCG trigger and oocyte vitrification?

ObjectiveOocyte vitrification has been developed as a promising alternative to slow freezing; however, the clinical outcome is highly operator dependent. From the past study, we know the timing of cryoprotectant exposure and understand that the intervals between the application of liquid nitrogen and thawing solution are crucial factors in the vitrification process. However, the optimal time intervals between hCG trigger and oocyte vitrification and equilibration remain unknown. This study aimed to evaluate the optimal intervals before and during modified vitrification.Materials and methodsThis retrospective study included 66 patients undergoing vitrified-thawed oocyte cycles from June 2018 to May 2019. Oocyte in vitro maturation (IVM) is defined as the maturation in vitro of an immature oocyte collected using a standard pick up procedure. Oocytes were grouped into the following intervals: (1) human chorionic gonadotropin (hCG) trigger to oocyte vitrification (<38 h; 38–39 h; >39 h; IVM) (2) oocyte equilibration time (<10 min; 10–12 min; 12–15 min). The vitrification and warming procedures were performed following the steps as shown in the Cryotec method.ResultsA total of 390 mature oocytes were vitrified with the Cryotec method. The survival rates were not significantly different among the various intervals after the hCG trigger (97.59%; 95.54%; 100%); however, there was a trend of decreased survival rate in IVM group (66.67%). The oocyte survival rates were not significantly different among the various times of oocyte equilibration (96.77%; 97.33%; 95.42%).ConclusionsThis was the first study to demonstrate no correlation between oocyte survival rate and the time intervals between hCG trigger and oocyte vitrification. Nor did the oocyte survival rate correlate with the various equilibration times during vitrification, as long as the oocyte was mature. In the future, large, prospective, randomized controlled studies will be required to confirm the clinical outcomes.     

Approaches to oocyte retrieval for advanced reproductive technology cycles planning to utilize in vitro maturation: a review of the many choices to be made

PurposeTo evaluate the minutiae associated with oocyte retrieval for use in human in vitro maturation IVF cycles. Many of the relevant features of oocyte retrieval were identified by the Trounson group in the first publication on successful in vitro maturation using transvaginal oocyte harvesting and these were a major focus of this review.MethodsPublished human and animal studies, together with topics from mathematics and mechanics, were used to try to understand the importance of different choices that could be made in structuring a transvaginal oocyte retrieval procedure in humans.ResultsThe published literature suggests that the highest oocyte recovery rate occurs using higher pressures and thicker needles, but this comes at the cost of damaging the cumulus oocyte complex. It is likely that this damage is caused by the sheer stress forces exerted on the cumulus oocyte complex due to parabolic forces associated with laminar flow within the needle and is likely worsened by irregular forces during intervals of turbulent flow occurring with entry into the needle. Larger needles also cause more pain and may be associated with more blood loss. Higher velocity entry into the follicle, needle rotation to prevent premature blockage of the lumen, and carefully timed applications of aspiration pressure theoretically optimize oocyte retrieval technique.ConclusionsOocyte retrieval for in vitro maturation is effected by the interaction of the many choices that need to be made in planning for the procedure. The most difficult decision involves aspiration pressure or fluid flow rate and needle size.     

Familial partial 17,20-desmolase and 17alpha-hydroxylase deficiency presenting as infertility

Purpose : Females with 17-hydroxylase/17,20-desmolase deficiency normally present with amenorrhea, sexual infantilism, hypertension, and hypokalemia. We report on a new clinical presentation of this combined enzymatic defect. Methods : Four Jewish women from two unrelated families presented with primary infertility. All patients exhibited a normal phenotype, blood pressure, and serum potassium levels, with abnormally high follicular phase serum progesterone and low E₂ levels. In order to characterize the underlying defect, the following steps were undertaken: 1) ovarian suppression by GnRH agonist, 2) adrenal suppression by dexamethasone, 3) ovarian stimulation by gonadotropins, 4) adrenal stimulation by ACTH, 5) hormonal assessment of follicular fluid aspirates, and 6) assessment of in vitro E₂ production by luteinized granulosa cells. Results : The clinical characteristics and endocrine testing results support the diagnosis of a partial deficiency in 17-hydroxylase/17,20-desmolase activities, shared by the adrenal gland and the ovaries. Conclusions : Female infertility can be the first and sole clinical manifestation of this enzymatic defect. Its exact nature and prevalence remain to be determined.     

Comprehensive elucidation of amino acid profile in human follicular fluid and plasma of in vitro fertilization patients

Abstract

The assessment of oocyte quality in human in vitro fertilization (IVF) is in focus of intensive studies. Follicular fluid (FF) constitutes the major medium for the developing oocyte and therefore it is a perfect target to search for the biomarkers of female infertility. The objective of this study was to compare the amino acid (AA) profile of human FF and plasma (PL) (from 96 IVF patients) and to examine if the AA composition is related to oocyte quality, IVF results and women’s infertility. In both biological fluids, Gln, Gly and Ala appeared as dominant AAs. Most of AAs are more abundant in PL; the exceptions are Glu, Thr, Ala and Gly, which are higher in FF and Gln, Arg and Phe, the contents of which are similar in both biological fluids. Ser in FF and Met and Phe in PL were detected as potential biomarkers as their content varied depending on the IVF outcome. Significant differences were also detected between the groups of different infertility reasons. Our results suggest that intra-follicular AAs might reflect the condition of the preovulatory follicle and together with PL, AAs can be used to characterize the infertility etiology and oocyte quality related to IVF outcome.     

Recovery and Maturation of Immature Oocytes in Patients at Risk for Ovarian Hyperstimulation Syndrome

Purpose: Our purpose was to examine the rate of immature oocyte recovery and their potential for in vitro maturation from canceled human menopausal gonadotropin cycles due to the risk of having ovarian hyperstimulation syndrome develop. Methods: Patients underwent ultrasound-guided immature oocyte pickup. The number of oocytes recovered from these patients was recorded, and then cultured in vitro. Cumulus expansion and the stage of nuclear maturation were observed after 24 and 48 hr, respectively. Results: Seventeen patients underwent 20 immature oocyte recoveries. A total of 162 oocytes (8.1 oocytes/patient) was obtained. All of the oocytes were enclosed in dense layers of cumulus cells. Among them, 78.4% showed cumulus expansion after 24 hr and 66% completed meiotic maturation to metaphase II after 48 hr in culture. There was only one immature oocyte pickup in which no oocytes were recovered (95% recovery rate). None of the patients had ovarian hyperstimulation syndrome develop. Conclusions: Immature oocytes can be recovered from canceled human menopausal gonadotmpin cycles in patients who are at potential risk for severe hyperstimulation syndrome. These oocytes can be matured in vitro and can be used for clinical and research purposes as well.     

Outcome of IVF in Patients with Endometriosis in Comparison with Tubal-Factor Infertility

Purpose: The aim of this retrospective study was to compare the outcome of in vitro fertilization and embryo transfer in women with endometriosis and a control group with tubal-factor infertility. Methods: Forty-eight patients with endometriosis underwent 65 cycles of in vitro fertilization and embryo transfer at Huddinge University Hospital. The matched control group with tubal-factor infertility consisted of 98 cycles in 98 patients. These groups were retrospectively analyzed regarding stimulation, fertilization, embryo development, implantation, and pregnancy outcome. Results: The fertilization rate was significantly lower in women with endometriosis, but the cleavage, implantation, and pregnancy rates did not differ. Conclusions: Our results show that women with endometriosis have a lower fertilization rate compared with women with tubal-factor infertility. However, once the oocyte is fertilized, it seems that the preembryo has a normal chance of implantation, leading to similar pregnancy rates.     

FSH dose is negatively correlated with number of oocytes retrieved: analysis of a data set with ~650,000 ART cycles that previously identified an inverse relationship between FSH dose and live birth rate

Purpose

To evaluate whether total FSH dose was negatively correlated with number of oocytes retrieved in a large data set where previously, a negative correlation between FSH dose and live birth rate was identified.

Methods

Data from 650,637 fresh autologous in vitro fertilization (IVF) cycles reported to the Society for Assisted Reproductive Technology between 2004 and 2012 were included. Logistic regression analysis was performed to determine if the relationship between total FSH dose used during ART with number of oocytes retrieved was impacted by the patient’s health prognosis, age, BMI, ovarian stimulation protocol, or infertility diagnosis.

Results

The number of oocytes retrieved was negatively correlated with FSH dose (P < 0.0001). Regardless of patient prognosis, age, BMI, ovarian stimulation protocol, and infertility diagnosis, the highest number of oocytes retrieved was in the 1001–2000 IU FSH group, and was 36–51% lower in the > 5000 IU compared with the optimal, 1001–2000 IU, FSH groups. Overall, ~80% of patients received FSH doses outside of the optimal FSH dose. Moreover, 61% of good prognosis patients (excludes individuals likely prescribed higher FSH doses) received doses exceeding the optimal dose range.

Conclusion

The inverse relationship between FSH dose and the number of oocytes retrieved independent of patient age or health implies that excessive FSH doses during ART may be detrimental to oocyte retrieval.