The human sperm basal body is a complex centrosome important for embryo preimplantation development.

The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis, has not been directly addressed so far. Using proteomics and immunofluorescence studies, we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetic activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar material (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

STUDY QUESTION: Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? SUMMARY ANSWER: New mutations in the PLCZ1 sequence are associated with FFs after ICSI. WHAT IS KNOWN ALREADY: FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. STUDY DESIGN, SIZE, DURATION: We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. MAIN RESULTS AND THE ROLE OF CHANCE: In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. WIDER IMPLICATIONS OF THE FINDINGS: PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

Are we ready to inject? Individualized LC-CUSUM training in ICSI.

PURPOSE: The objective of this prospective, single center study was to develop a personalized training scheme for intracytoplasmic sperm injection (ICSI) through the use of learning curve-cumulative summation (LC-CUSUM), which allows to tailor training to the trainee performance, and to validate it against the performance of experienced embryologists. METHODS: Five trainees microinjected latex microspheres (LM) into vitro matured oocytes. A microinjection was considered successful when the oocyte did not lyse in the 24 h following the injection. RESULTS: Each trainee became proficient at ICSI after a variable number of injections, ranging from 35 to 80. Trainees that achieved proficiency went on to perform ICSI with human gametes in a clinical setting with proficiency comparable to that of experienced embryologists. CONCLUSIONS: We show that LC-CUSUM based personalized ICSI training is feasible and allows trainees to be as proficient as trained embryologists when treating actual patients.

PLCζ disruption with complete fertilization failure in normozoospermia.

PURPOSE: Intracytoplasmic sperm injection (ICSI) is widely used to achieve fertilization in the presence of severe male factor, resulting in high fertilization rates. Nevertheless, 1-3 % of couples experience complete fertilization failure after ICSI. When a male factor is identified, assisted oocyte activation (AOA) can help overcome fertilization failures. The objective of this study is to describe a case of repeated complete fertilization failures after ICSI with donor oocytes, and to investigate the molecular and functional aspects of phospholipase C zeta (PLCζ) protein in the patient semen. METHODS: The patient was a normozoospermic male who had previously fathered, through natural conception, four children by a different partner. Molecular and functional analysis of sperm-specific PLCζ in the patient and control samples by means of gene sequencing, immunocytochemistry, Western blot, mouse oocyte activation test (MOAT), and mouse oocyte calcium analysis (MOCA) were used. RESULTS: PLCζ expression levels and distribution were significantly disrupted, although MOAT and MOCA did not indicate a decrease in activation ability. CONCLUSIONS: Normozoospermic males can have disrupted expression and distribution of PLCζ, and reduced activation ability after ICSI in human oocytes, despite their normal activation potential in functional testing using mouse oocytes. Discrepancy among molecular and functional data might exist, as mutations in the gene sequence may not be the only cause of alteration in PLCζ protein related to activation failures.

Mitochondrial DNA depletion in oocytes of HIV-infected antiretroviral-treated infertile women.

BACKGROUND: HIV-infected women under highly active antiretroviral therapy (HAART) undergoing in vitro fertilization (IVF) have a lower pregnancy rate than noninfected controls, which depends on oocyte-related factors. We hypothesized that mitochondrial toxicity caused by antiretrovirals could be the underlying mechanism of such disturbance. METHODS: We have studied 16 and 19 frozen-thawed oocytes obtained after oocyte retrieval IVF cycles from 8 and 14 infertile HIV-infected and uninfected women, respectively, matched by age. At inclusion, HIV-positive women had been infected for >13 years and had received HAART for >9 years, including at least one nucleoside reverse transcriptase inhibitor. All of them had undetectable HIV viral load and a good immunological status. Mitochondrial DNA (mtDNA) content was determined by quantitative real-time PCR in each individual oocyte. RESULTS: HIV-infected infertile women on HAART showed significant oocyte mtDNA depletion when compared with uninfected controls (32% mtDNA decrease, P<0.05). This oocyte mtDNA depletion was even greater on those HIV-infected women who failed to become pregnant when compared with controls (39% mtDNA decrease, P=0.03). No significant correlation was found between mtDNA oocyte content and cumulative doses of antiretrovirals or the immunological status of HIV patients. CONCLUSIONS: Oocytes from infertile HIV-infected HAART-treated women show decreased mtDNA content, and this could explain their poor reproductive outcome.

Endometrial receptivity after oocyte donation in recipients with a history of chemotherapy and/or radiotherapy.

INTRODUCTION: Information is scarce regarding the outcome of oocyte donation (OD) in patients with a history of cancer treatment. Therefore, we conducted a matched controlled analysis on the outcome of OD in these recipients. METHODS: Between January 2000 and November 2005, 33 patients with a history of chemotherapy and/or radiotherapy had an OD cycle. Matching was performed to the chronologically closest patient without a history of cancer therapy by number of days of hormonal stimulation before embryo replacement, number of replaced embryos, day of embryo transfer and origin of sperm. RESULTS: The primary diseases of the patients were Hodgkin's lymphoma (n = 12), non-Hodgkin's lymphoma (n = 3), leukaemia (n = 7), ovarian cancer (n = 6), Ewing's sarcoma (n = 2), breast cancer (n = 1), sympathoblastoma (n = 1) and histiocytosis X (n = 1). Twenty-three patients had undergone chemotherapy and radiotherapy, nine patients chemotherapy only and one radiotherapy only. The mean age of the recipients was 33.1 years [95% confidence interval (CI) 30.9-35.3] and 39.6 (95% CI 37.1-42.1) in the study and control groups, respectively. The average number of received oocytes and transferred embryos, was similar in both groups. Nineteen (57.6%) versus 13 (39.4%) pregnancies resulting in an ongoing pregnancy (i.e. viable at 12 weeks) in 15 (45.4%) versus 9 cycles (27.3%) (NS) were obtained in study and control groups, respectively. Implantation rate in study and control groups was 35.8 versus 17.9%, respectively (P = 0.02). CONCLUSIONS: The results suggest that patients with a history of cancer treatment have a pregnancy rate after OD similar to that in the general population of oocyte recipients.

Prognostic factors in oocyte donation: an analysis through egg-sharing recipient pairs showing a discordant outcome.

OBJECTIVE: To analyze prognostic factors that are associated with a discordant outcome in egg recipients sharing oocytes from the same donor. DESIGN: Matched case-control single-center study. SETTING: Private infertility clinic. PATIENT(S): Four hundred forty-four recipients (222 pairs) sharing oocytes from the same donor and showing a discordant outcome. INTERVENTION(S): Controlled ovarian hyperstimulation of egg donors, oocyte donation, intracytoplasmic sperm injection, and ET in egg recipients. MAIN OUTCOME MEASURE(S): Recipient age, obstetric (gravidity, parity) and gynecologic variables (previous uterine surgery, uterine fibroids, uterine malformations, endometriosis, history of tubal infertility), previous oocyte donation cycles, duration of E(2) replacement, received cumulus-oocyte complexes, mature (MII) oocytes, fertilized oocytes, transferred embryos, mean embryo score, transfer difficulty, and semen parameters. RESULT(S): No significant differences were found in the above-mentioned prognostic factors between the study and control groups. CONCLUSION(S): Recipient- and cycle-related prognostic factors investigated in our study were not associated with a discordant outcome in recipient pairs sharing oocytes from the same donor. Other possible prognostic factors involving oocyte donor heterogeneity, embryo aneuploidy rates, male factor infertility, and endometrial receptivity should be further investigated.

Decreased pregnancy rate after in-vitro fertilization in HIV-infected women receiving HAART.

A study on in-vitro fertilization (IVF) was conducted among HIV-infected women. In these patients, a reduced pregnancy rate after IVF was observed if the patient's own oocytes were used. However, no significant reduction in the pregnancy rate was found if donated oocytes were used. The CD4 lymphocyte count was independently associated with ovarian resistance to hyperstimulation. Subclinical hypogonadism mediated by immunosuppression may explain these observations, suggesting the need to optimize the immunological status of the patient before considering assisted reproduction treatments.

Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies.

We used fluorescent in situ hybridisation (FISH) to detect nine chromosomes (1, 13, 15, 16, 17, 18, 21, 22 and X) in 89 first Polar Bodies (1PBs), from in vitro matured oocytes discarded from IVF cycles. In 54 1PBs, we also analysed the corresponding oocyte in metaphase II (MII) to confirm the results; the other 35 1PBs were analysed alone as when preimplantation genetic diagnosis using 1PB (PGD-1PB) is performed. The frequency of aneuploid oocytes found was 47.5%; if the risk of aneuploidy for 23 chromosomes is estimated, the percentage rises to 57.2%. Missing chromosomes or chromatids found in 1PBs of 1PB/MII doublets were confirmed by MII results in 74.2%, indicating that only 25.8% of them were artefactual. Abnormalities observed in 1PBs were 55.8% whole-chromosome alterations and 44.2% chromatid anomalies. We observed a balanced predivision of chromatids for all chromosomes analysed. Differences between balanced predivision in 1PB and MII were statistically significant (P&<0.0001, chi(2) test); the 1PB was most affected. The mean abnormal segregation frequency for each chromosome was 0.89% (range 0.52-1.70%); so, each of the 23 chromosomes of an oocyte has a risk of 0.89% to be involved in aneuploidy. No significant differences were observed regarding age, type of abnormality (chromosome or chromatid alterations) or frequency of aneuploidy. Nine of the 35 patients (25.7%) whose 1PB and MII were studied presented abnormalities (extra chromosomes) that probably originated in early oogenesis. Analysis of 1PBs to select euploid oocytes could help patients of advanced age undergoing in vitro fertilization (IVF) treatment.