Assessment of cell-free DNA and apoptosis in an oocyte microenvironment: promising biomarkers to predict intracytoplasmic sperm injection outcomes.

Cell-free DNA (cf-DNA) is defined as DNA fragments that are released into the body fluids from apoptosis or necrosis cells, including follicular fluid (FF), which can affect the microenvironment of the oocyte associated with infertility. We aimed to investigate a relationship between apoptosis of cumulus cells (CCs) and cf-DNA levels in FF and clinical outcomes of women undergoing intracytoplasmic sperm injection (ICSI). Therefore, 82 FF samples were collected, and the corresponding CCs were isolated for ICSI procedures. FF cf-DNA concentration was quantified using ALU-quantitative polymerase chain reaction (PCR), and CCs DNA fragmentation index (DFI) was evaluated by the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) method. We found that cf-DNA and DFI levels were significantly higher in FF and CCs samples related to the age of women ≥37 years compared with the age of women < 37 years. Moreover, in older and younger women, FF cf-DNA and CCs DFI levels were significantly lower when the anti-Müllerian hormone (AMH) level was > 1.1 ng/ml compared with when AMH ≤ 1.1 ng/ml. In addition, patients with a low number of retrieved oocytes ≤ 6 had significantly higher levels of CCs DFI and FF cf-DNA than women with a higher number of retrieved oocytes > 6. Additionally, we observed that higher levels of cf-DNA and DFI were associated with poor oocyte maturity and poor embryo quality. Finally, cf-DNA and DFI levels were significantly lower in pregnant women than in non-pregnant ones. We conclude that DFI and cf-DNA levels in the oocyte microenvironment could have potential use in evaluating oocyte and embryo developmental competence.

Influence of endometrioma size on ART outcomes.

RESEARCH QUESTION: Does endometrioma size affect the number of oocytes retrieved after ovarian stimulation in women with endometriosis-related infertility undergoing IVF/intracytoplasmic sperm injection (ICSI)? DESIGN: Cohort study of infertile women with unilateral or bilateral endometrioma(s) associated with deep infiltrating endometriosis, undergoing their first IVF/ICSI cycle between January 2014 and November 2021. A total of 326 women with an adequate imaging work-up with transvaginal ultrasound and/or magnetic resonance imaging performed by senior radiologists before the start of ovarian stimulation was included. Prognostic factors associated with the number of oocytes retrieved were analysed. IVF/ICSI outcomes were compared between five groups defined according to the largest endometrioma diameter (<2, 2 to <4, 4 to <6, 6 to <8 and ≥8 cm). RESULTS: Factors that significantly reduced the number of oocytes retrieved after adjustment by multiple linear regression were women's age (regression coefficient -0.18; 95% confidence interval [95% CI] -0.31 to-0.06; P = 0.005), smoking habit (-2.02; 95% CI -3.42 to -0.62; P = 0.005), day 3 FSH concentration (-0.20; 95% CI -0.39 to -0.02; P = 0.031) and a previous history of surgery for ovarian endometriosis (-1.32; 95% CI -2.63 to -0.02; P = 0.047). Antral follicle count and oestradiol concentration on the trigger day were positively correlated with the number of oocytes retrieved (0.14; 95% CI 0.08 to 0.19; P < 0.001 and 0.003; 95% CI 0.002 to 0.004; P < 0.001, respectively). The mean number of oocytes retrieved was not significantly different between the five groups (P = 0.413), nor were the cumulative live birth rate, the number of cancelled cycles and perinatal outcomes. CONCLUSIONS: No significant difference in the number of oocytes retrieved was observed according to endometrioma size. This study suggests that ovarian stimulation can be of benefit to women irrespective of the endometrioma size.

Sperm Chromatin Condensation Defect Accelerates the Kinetics of Early Embryonic Development but Does Not Modify ICSI Outcome.

The origin and quality of gametes are likely to influence the kinetics of embryonic development. The purpose of the study was to assess the impact of sperm nuclear quality, and in particular sperm chromatin condensation, on the kinetics of early embryo development after intracytoplasmic sperm injection (ICSI). Our study included 157 couples who benefitted from ICSI for male factor infertility. Chromatin condensation and DNA fragmentation were assessed in spermatozoa prior to ICSI. Above the 20% threshold of sperm condensation defect, patients were included in the abnormal sperm chromatin condensation (ASCC) group; below the 20% threshold, patients were included in the normal sperm chromatin condensation (NSCC) group. After ICSI, the oocytes were placed in the time-lapse incubator. The kinetics of the cohort's embryonic development have been modeled. The fading times of pronuclei and the time to two blastomeres (t2, first cleavage) and four blastomeres (t4, third cleavage) differed significantly between the NSCC and ASCC groups, with earlier events occurring in the ASCC group. On the other hand, the state of sperm chromatin condensation did not seem to have an impact on live birth rates or the occurrence of miscarriages. The kinetics of early embryonic development was accelerated in males with a sperm chromatin condensation defect without compromising the chances of pregnancy or promoting miscarriage. However, our study highlights the paternal contribution to early embryonic events and potentially to the future health of the conceptus.

Patients with acephalic spermatozoa syndrome linked to novel TSGA10/PMFBP1 variants have favorable pregnancy outcomes from intracytoplasmic sperm injection.

Acephalic spermatozoa syndrome is a rare form of teratozoospermia characterized by headless spermatozoa. Previous studies have found that variants in SUN5, PMFBP1, TSGA10, BRDT, and SPATC1L are associated with this phenotype. Many researchers have suggested that variants in TSGA10 without a proximal centriole might influence early embryonic development. This retrospective cohort study included 12 infertile men with severe acephalic spermatozoa in China. We identified six heterozygous variants and four homozygous variants in TSGA10/PMFBP1 in seven patients by whole-exome sequencing (WES). Acephalic spermatozoa defects due to different genetic variations may affect only spermatozoa morphology but do not reduce the chances of fertilization, affect embryo quality at early stages or impair intracytoplasmic sperm injection (ICSI) outcomes. Patients with TSGA10/PMFBP1 variations were all expected to have good prognoses with ICSI.

Occurrence of smooth endoplasmic reticulum aggregates in metaphase II oocytes: relationship with stimulation protocols and outcome of ICSI and IVF cycles.

STUDY QUESTION: Is there any association between the appearance of smooth endoplasmic reticulum aggregates (SERa) in oocytes and ovarian stimulation, embryological, clinical and neonatal outcomes of ICSI and IVF cycles? SUMMARY ANSWER: A suboptimal prolonged ovarian stimulation is detrimental to oocytes by inducing the occurrence of SERa, which reduces the reproductive potential of oocytes. WHAT IS KNOWN ALREADY: Controlled ovarian stimulation recruits oocytes of different qualities. Based on current evidence, it was agreed that non-homogeneous cytoplasm may represent the normal variability among oocytes rather than a dysmorphism with developmental significance. The only exception is the appearance of SERa within the ooplasm. Owing to the lack of univocal evidence in this literature about the safety of injecting oocytes with SERa and the mechanism responsible for the occurrence of SERa, this topic is still a matter of debate. STUDY DESIGN, SIZE, DURATION: A retrospective, longitudinal cohort study performed at a tertiary level public infertility center. We included 1662 cycles (180 SERa+ and 1482 SERa-) from 1129 women (age: 20-44 years) who underwent IVF/ICSI treatments in 2012-2019. The SERa+ cycles had at least one SERa+ oocyte in the oocyte cohort. The SERa- cycles had morphologically unaffected oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected stimulation data and embryological, clinical, neonatal outcomes of SERa- and SERa+ cycles and oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 347 out of 12 436 metaphase II oocytes (2.8%) were affected by SER. We performed only 12 transfers involving at least one SERa+ embryo. Stimulation length (P = 0.002), serum progesterone (P = 0.004) and follicle size (P = 0.046) at trigger, number of retrieved (P = 0.004) and metaphase II (P = 0.0001) oocytes were significantly higher in SERa+ than SERa- cycles. Fertilization rate was significantly (P < 0.0001) reduced in SERa+ cycles and oocytes compared to SERa- counterparts. Embryos of SERa+ cycles had a lower blastocyst formation rate compared to embryos of SERa- cycles (P = 0.059). Statistical analysis according to a generalized estimating equation model performed at patient level demonstrated that the duration of ovarian stimulation was predictive of SERa+ oocytes appearance. The clinical success of SERa+ cycles was lower than SERa- cycles, although no differences in neonatal birthweights or malformations were recorded in sibling unaffected oocytes of SERa+ cycles. LIMITATIONS, REASONS FOR CAUTION: Given that SERa+ oocytes were discarded in our center for years and transfers of embryos originating from affected oocytes were generally avoided, clinical outcomes of SERa+ cycles are largely attributable to the transfer of embryos derived from unaffected oocytes of SERa+ cycles and we did not have data about newborns from affected oocytes, since none of the transfers involving SERa+ embryos resulted in a progressive clinical pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, we speculate that the late-follicular phase elevated serum progesterone caused by a suboptimal prolonged ovarian stimulation may be detrimental to the oocytes by inducing the occurrence of SERa, resulting in negative effects on their reproductive potential. This raises the question of whether some stimulation regimens could be worse than others and a change in stimulation protocol would reduce the possibility of producing oocytes with suboptimal maturation. In particular, our data highlight the importance of correct timing of the trigger in order to maximize oocyte collection, not only in terms of numerosity but also their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Two frequent loss-of-function mutations in Aurora Kinase C gene in Algerian infertile men with macrozoospermia.

Macrozoospermia is associated with severe male infertility. To date, the only gene implicated in this phenotype is the Aurora Kinase C gene. We report in this work the genetic screening of AURKC mutations in 34 patients with macrozoospermia among 3,536 Algerian infertile men. Nineteen patients (56%) were homozygotes for the c.144delC mutation, eight (23.52%) homozygotes for the c.744C>G (p.Y248*) mutation and two (5.88%) compound heterozygotes. No AURKC mutation was identified in five patients (14.7%). Interestingly and although it is generally accepted that nearly all positive mutated AURKC patients have close to 100% large-head spermatozoa, our results showed that 11 patients with AURKC mutations (32.35%) had large-headed spermatozoa lower than 70% (7 with c.144delC and 4 with p.Y248*), and no mutation was found in 2 patients who had >70% of macrocephalic spermatozoa. Twenty ICSI attempts were performed before genetic screening resulting in 39 embryos but no pregnancy was obtained. The sequencing of AURKC exons 3 and 6 is appropriate as a first-line genetic exploration in these patients to avoid unsuccessful ICSI attempts. A percentage of large head spermatozoa beyond 25% and a percentage of multiflagellar spermatozoa beyond 10% are predictive of a positive mutation diagnosis.

Male ageing is negatively associated with the chance of live birth in IVF/ICSI cycles for idiopathic infertility.

STUDY QUESTION: Is male age associated with the clinical outcomes of IVF/ICSI cycles for idiopathic infertility after adjustment for female age? SUMMARY ANSWER: Male ageing is negatively associated with clinical IVF/ICSI outcomes in couples with idiopathic infertility independent of female age. WHAT IS KNOWN ALREADY: The effect of male age on the outcomes of infertility treatments is controversial and poorly explored. In contrast, fertility is known to decline significantly with female age beyond the mid-30s, and reduced oocyte quality plays an important role. The negative effect of male age on sperm quality is largely associated with an increasing susceptibility to sperm DNA damage. Although increasing maternal age has been linked with poorer oocyte quality, studies on the effect of male age have disregarded the need to control for female age making it difficult to define clearly the role of male age in infertile couples. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study analysed 2425 cycles of couples with idiopathic infertility selected from a total of 24 411 IVF/ICSI cycles performed at Monash IVF in Australia between 1992 and 2017. The primary outcome was live birth and secondary outcomes were clinical pregnancy and miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples with primary/secondary infertility who underwent IVF/ICSI cycles with male partners classified as normozoospermic were selected (inclusion criteria). Couples in which the female partner had endometriosis, tubal factors, polycystic ovarian syndrome, ovarian hyperstimulation syndrome, poor responders (≤3 mature oocytes retrieved) and couples with more than 15 cumulus oocyte complexes retrieved or who used cryopreserved gametes were excluded. Binary logistic multilevel modelling was used to identify the effect of male age and female age on clinical outcomes after controlling for confounding factors. Male age and female age were examined as continuous and categorical (male age: <40, 40-44, 45-49, 50-54, ≥55; female age:<30, 30-34, 35-39, ≥40) predictors. MAIN RESULTS AND THE ROLE OF CHANCE: There was a negative effect of male age and female age on live birth as odds ratios (OR) with 95% CI for each additional year of age (OR-male age: 0.96 [0.94-0.98]; OR-female age: 0.90 [0.88-0.93] P < 0.001). Potential interactions with male age such as type of treatment (IVF/ICSI), embryo transfer day (Day 3/Day 5) and female age did not have significant associations with outcomes (P > 0.05). Secondary outcomes showed a significant reduction in the odds of clinical pregnancy (OR-male age: 0.97 [0.96-0.99]; OR-female age: 0.92 [0.89-0.94] P < 0.001) and an increase in the odds of miscarriage with older age: male age (OR: 1.05 [1.01-1.08]; P = 0.002); female age (OR: 1.11 [1.05-1.18]; P < 0.001). Worse outcomes were associated with more cycles (clinical pregnancy-OR: 0.96 [0.93-0.99] P = 0.03; live birth-OR: 0.96 [0.92-0.99] P = 0.023) while more inseminated oocytes were associated with better outcomes (clinical pregnancy-OR: 1.06 [1.03-1.06] P < 0.001; live birth-OR: 1.07 [1.04-1.11] P < 0.001). Analyses for age categories showed a gradual worsening of clinical outcomes with increasing male age, with a significantly worse live birth and clinical pregnancy outcomes in males aged older than 50 years compared to males younger than 40 years (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study is limited to the information on confounding factors included. The study may also be limited in its generalizability to a wider population due the strict selection criteria. Age as a category could potentially result in residual confounding due to categorizing a continuous variable. WIDER IMPLICATIONS OF THE FINDINGS: This study provides information for counselling of couples with idiopathic infertility. STUDY FUNDING/COMPETING INTEREST(S): Funded by the Education Program in Reproduction and Development, Department of Obstetrics and Gynaecology, Monash University. None of the authors has any conflict of interest to report. TRIAL REGISTRATION NUMBER: N/A.

Novel use of COMET parameters of sperm DNA damage may increase its utility to diagnose male infertility and predict live births following both IVF and ICSI.

STUDY QUESTION: Do the Comet parameters of the proportions of sperm with low or high DNA damage improve the power of the test in the diagnosis of male infertility and/or prediction of IVF and ICSI live birth rates? SUMMARY ANSWER: The mean Comet score and the scores for proportions of sperm with high or low DNA damage were useful in diagnosing male infertility and provided additional discriminatory information for the prediction of both IVF and ICSI live births. WHAT IS KNOWN ALREADY: Sperm DNA damage impacts adversely on male fertility and IVF outcomes. STUDY DESIGN, SIZE, DURATION: A retrospective study was performed involving a total of 457 participants (381 patients and 76 fertile donors). Data was collected from a fertility clinic between 2015 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 381 consecutive male partners of couples attending for ART and 76 fertile donors were included in the study. DNA fragmentation was measured by the alkaline Comet assay. Receiver operator characteristic curve analysis (area under the ROC curve (AUC)) was used to determine the value of average Comet score (ACS), low Comet score (LCS) and high Comet score (HCS) to diagnose male factor infertility. In total, 77 IVF and 226 ICSI cycles were included to determine thresholds for each parameter (AUC analysis) and to compare live birth rates (LBRs) following each ART. MAIN RESULTS AND THE ROLE OF CHANCE: ACS, HCS and LCS were predictive of male infertility (AUC > 0.9, P < 0.0001). IVF LBRs declined once DNA damage exceeded the threshold levels. HCS showed the sharpest decline. Following ICSI, the highest LBRs were in men whose DNA damage levels approached the fertile range. Trends differed in IVF. LBRs decreased as damage increased whereas in ICSI the LBRs decreased but then remained stable. LIMITATIONS, REASONS FOR CAUTION: Since this is the first study to show the impact of sperm DNA damage on ICSI live births, a prospective study should be performed (stratifying patients to IVF or ICSI based on these thresholds) to validate this study. WIDER IMPLICATIONS OF THE FINDINGS: Our study presents novel information towards elucidating the genetic basis of male infertility and secondly on relevance of the extent of DNA damage as an impending factor in both IVF and ICSI success. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Examenlab Ltd, The Lister Clinic, Cryos International and Imperial College London NHS Trust. No external funding was obtained for this study. SL and KL are employees of Examenlab Ltd, a university spin-out company with a commercial interest in sperm DNA damage. No other author has a conflict of interest to declare. TRIAL REGISTRATION NUMBER: Non-applicable.

Sperm chromatin condensation defects, but neither DNA fragmentation nor aneuploidy, are an independent predictor of clinical pregnancy after intracytoplasmic sperm injection.

PURPOSE: The impact of sperm DNA damage on intracytoplasmic sperm injection (ICSI) outcomes remains controversial. The purpose of the study was to evaluate the prognostic value of several types of sperm nuclear damage on ICSI clinical pregnancy. METHODS: Our retrospective study included a total of 132 couples who consulted for male or mixed-factor infertility that benefited from ICSI cycles from January 2006 to December 2015. All infertile males presented at least one conventional semen parameter alteration. Sperm nuclear damage was assessed using the Motile Sperm Organelle Morphological Examination for sperm head relative vacuolar area (RVA), aniline blue staining for chromatin condensation, terminal deoxynucleotidyl transferase dUTP nick-end labeling for DNA fragmentation, and fluorescence in situ hybridization for aneuploidy. RESULTS: Infertile males who achieved pregnancy after ICSI had fewer chromatin condensation defects than did males who did not achieve any pregnancy (15.8 ± 12.0% vs. 11.4 ± 7.9%, respectively, P = 0.0242), which remained significant in multivariate regression analysis (RR = 0.40 [0.18 to 0.86], P = 0.02). RVA, DNA fragmentation, and aneuploidy were not predictive factors of ICSI outcomes. The pregnancy rate was significantly decreased by number of progressive motile spermatozoa with normal morphology after migration (P = 0.04). In female partners, 17ß estradiol of less than 2000 pg/mL on the day of ovulation induction significantly reduced the occurrence of clinical pregnancy (P = 0.04). CONCLUSION: Sperm chromatin condensation defects were more frequently observed in couples with ICSI failure and should be considered a negative predictive factor for the occurrence of clinical pregnancy.

An adapted carrier for the cryopreservation of human testicular spermatozoa.

RESEARCH QUESTION: Does the adapted carrier Cryoplus improve the quality of cryopreserved spermatozoa compared with the use of conventional containers, and what is the effect of the adapted carrier on clinical outcomes? DESIGN: Semen samples from 27 cases of oligozoospermia were used to investigate whether the adapted carrier improved cryopreserved sperm quality compared with the use of 0.25-ml straws and 2-ml cryogenic vials. Thirty testicular sperm samples were used to study the quality of testicular spermatozoa cryopreserved in the adapted carrier. The retrospective study included a further 104 men with azoospermia to investigate the clinical outcomes of testicular spermatozoa cryopreserved with the adapted carriers. Men with mostly obstructive azoospermia were included in this study. RESULTS: The adapted carrier improved cryopreserved spermatozoa motility of semen samples compared with 2-ml cryogenic vials but not compared with 0.25-ml straws. No differences were found in cryopreserved sperm DNA fragmentation among the three carriers. Fertilization and good-quality embryo rates were similar in ICSI cycles using fresh or cryopreserved testicular spermatozoa. Additionally, no difference was evident between frozen-thawed embryo transfer cycles using fresh or cryopreserved testicular spermatozoa in clinical pregnancy, implantation, miscarriage, live birth rates or birth weight. CONCLUSIONS: The adapted carrier improved the cryopreserved sperm motility compared with the effects of 2-ml cryogenic vials. The outcomes of intracytoplasmic sperm injection and frozen-thawed embryo transfer outcomes indicate that testicular spermatozoa cryopreserved using the adapted carrier is not inferior to fresh testicular spermatozoa. The use of the adapted carrier for cryopreserving human testicular spermatozoa especially from obstructive azoospermia is simple and effective.

Combined GnRH-agonist and human chorionic gonadotropin trigger improves ICSI cycle outcomes in patients with history of poor fertilization.

PURPOSE: The purpose of this study was to investigate the utility of a combined GnRH-agonist (GnRH-a) and human chorionic gonadotropin (hCG) trigger in improving ICSI cycle outcomes in patients with poor fertilization history after standard hCG trigger in prior ICSI cycles. METHODS: Retrospective cohort study. Patients with a fertilization rate of <20% in at least two prior ICSI cycles who subsequently underwent another ICSI cycle with hCG trigger were compared to those who underwent another ICSI cycle with a combined GnRH-a and hCG trigger. Oocyte maturity, fertilization, clinical pregnancy, and live birth rates were compared. A multiple linear regression model was used to explore the association between combined GnRH-a and hCG trigger (vs hCG trigger alone) and fertilization rate. RESULTS: A total of 427 patients with mean age of 37.3 ± 1.94 years and mean baseline fertilization rate of 17.9 ± 2.03% were included, of which 318 (74.5%) and 109 (25.5%) patients underwent a subsequent ICSI cycle with hCG and combined GnRH-a and hCG trigger, respectively. The baseline parameters of the male and female partner were similar. The mean fertilization rate in the combined trigger group was 16.4% (95% CI: 7.58-25.2%) higher than the hCG trigger group, even after adjustment for confounders. Patients in the combined trigger group had higher oocyte maturity (82.1 vs 69.8%), higher clinical pregnancy (27.5 vs 5.67%), and higher live birth rates (20.2 vs 3.46%) compared to the hCG trigger group. CONCLUSIONS: Combined GnRH-a and hCG trigger in ICSI cycles increase oocyte maturity, fertilization, clinical pregnancy, and live birth rates in patients with a history of poor fertilization after standard hCG trigger alone.

Does advanced paternal age affect global DNA methylation of human spermatozoa and intracytoplasmic sperm injection outcome?

Objective: This study was performed to (I) evaluate the potential effect of advanced paternal age on global DNA methylation in spermatozoa, and (II) to investigate the association between the outcome of intracytoplasmic sperm injection (ICSI), semen parameters, and advanced paternal age. Material and Methods: This study comprised 230 semen samples collected from males with a mean age of 38.2±8.5 years. Medical records were used to gather clinical information related to the female partner. The participants were divided into three groups depending on age: age <30 years; age 30-40 years; and age >40 years. The DNA was extracted from purified spermatozoa. Then the sperm global DNA methylation, sperm DNA fragmentation, and chromatin decondensation were evaluated by an ELISA, TUNEL, and Chromomycin A3 staining, respectively. Results: The sample counts were n=50 (21.8%), n=90 (39.1%) and n=90 (39.1%) for the <30, 30-40 and >40 year age-groups, respectively. A significant variation was found in the age of males included in this study (p<0.001). There was a significant reduction in sperm count, total motility, and non-progressive motility in the older group compared to the younger group (p<0.001). There was also a significant elevation in chromatin decondensation, DNA fragmentation, and global DNA methylation of spermatozoa in the older age group (p<0.001). Finally, there was a significant positive correlation between the percentage of non-motile sperm, sperm chromatin decondensation, DNA fragmentation, global DNA methylation status, and paternal age (p<0.001). Conclusion: These results suggest that advanced paternal age increased the DNA fragmentation, chromatin decondensation, and global DNA methylation level in human spermatozoa, which negatively affects the ICSI outcomes in couples undergoing ICSI cycles.

The impact of semen parameters on ICSI and pregnancy outcomes in egg recipient cycles with PGT-A.

BACKGROUND: The egg donation model offers an opportunity to isolate the male factor and evaluate its impact on IVF-intracytoplasmic sperm injection and pregnancy outcomes. OBJECTIVE: To study the effect of non-obstructive azoospermia on intracytoplasmic sperm injection and pregnancy outcomes compared with severe oligozoospermia and mild-to-moderate oligozoospermia in egg recipient cycles. MATERIALS AND METHODS: This is a retrospective longitudinal cohort study, including 1594 patients who underwent intracytoplasmic sperm injection in egg recipient cycles with preimplantation genetic testing for aneuploidies. The cohort was divided into three groups: couples with non-obstructive azoospermia accounting for 479 patients (30%); couples with severe oligozoospermia (sperm number <5 × 106 /mL), accounting for 442 patients (27.8%); couples with mild-to-moderate oligozoospermia, with sperm number >5 × 106 and <15 × 106 /mL, accounting for 673 patients (42.2%). RESULTS: The fertilisation rate was significantly reduced in the non-obstructive azoospermia group as compared with the severe oligozoospermia and the mild-to-moderate oligozoospermia group: 30.3% versus 63% and 77.3% (p < 0.05). Logistic regression analysis adjusted for confounders highlighted non-obstructive azoospermia as a negative predictor of obtaining a euploid blastocyst both per injected oocyte and per obtained blastocyst. The miscarriage rate in the non-obstructive azoospermia group was 11.8%; higher than the severe oligozoospermia and mild-to-moderate oligozoospermia groups (7% and 2.7%) (p < 0.05). The live birth rate per embryo transfer (ET) was significantly lower in the non-obstructive azoospermia group compared with the severe oligozoospermia and the mild-to-moderate oligozoospermia group (20.4% vs. 30.3% and 35.4%, p < 0.05). The risk of preterm labour was significantly higher in the non-obstructive azoospermia group, compared with the severe oligozoospermia and mild-to-moderate oligozoospermia group (55.1% vs. 46.8% and 16.1%, p < 0.001), and this difference was observed in both singleton and twin pregnancies. DISCUSSION AND CONCLUSION: In our retrospective comparative study, non-obstructive azoospermia significantly affects early embryonic potential and live birth rates per cycle and per embryo transfer. It is also associated with higher risk of preterm birth. Future prospective multi-centre studies are needed to highlight the effect of sperm quality on ART and pregnancy outcomes.

Inflammation mediates the effect of adiposity and lipid metabolism indicators on the embryogenesis of PCOS women undergoing in vitro fertilization/intracytoplasmic sperm injection.

Background: Polycystic ovary syndrome (PCOS) is a complex reproductive endocrine and metabolic disease affecting women of reproductive age. The low-grade chronic inflammation in PCOS is considered to be associated with obesity and dyslipidemia. We aim to investigate the potential mediating role of white blood cell (WBC) count, a representative inflammatory marker, in the effect of adiposity and lipid metabolism indicators on IVF/ICSI outcomes in PCOS women. Methods: We conducted a retrospective cohort study of 1,534 PCOS women who underwent their first IVF/ICSI cycles with autologous oocytes at a reproductive center from January 2018 to December 2020. The associations between PCOS women's adiposity and lipid metabolism indicators and WBC count and IVF/ICSI outcomes were examined using multivariable generalized linear models. Mediation analyses were conducted to evaluate the possible mediating role of WBC count. Results: We found significant dose-dependent correlations between adiposity and lipid metabolism indicators and IVF/ICSI outcomes (i.e., hormone levels on the ovulatory triggering day, oocyte development outcomes, fertilization, early embryo development outcomes, and pregnancy outcomes) (all p < 0.05), as well as between adiposity and lipid metabolism indicators and WBC count (all p < 0.001). Increasing WBC count was associated with adverse oocyte and embryonic development outcomes (all p < 0.05). Mediation analyses suggested that increasing serum TG and LDL-C levels and decreasing serum HDL-C level were significantly associated with reduced high-quality Day 3 embryo count in PCOS women, with 21.51%, 9.75%, and 14.10% mediated by WBC count, respectively (all p < 0.05). Conclusions: We observed significant associations between lipid metabolism indicators and high-quality Day 3 embryo count in PCOS women, partially mediated by inflammation-related mechanisms, suggesting the potential intervention target for improving embryo quality in PCOS women.

Adiposity and lipid metabolism indicators mediate the adverse effect of glucose metabolism indicators on oogenesis and embryogenesis in PCOS women undergoing IVF/ICSI cycles.

BACKGROUND: Polycystic ovary syndrome (PCOS) women have high incidences of dyslipidemia, obesity, impaired glucose tolerance (IGT), diabetes, and insulin resistance (IR) and are fragile to female infertility. Obesity and dyslipidemia may be the intermediate biological mechanism for the associations between glucose metabolism dysfunction and abnormal oogenesis and embryogenesis. METHODS: This retrospective cohort study was performed at a university-affiliated reproductive center. A total of 917 PCOS women aged between 20 and 45 undergoing their first IVF/ICSI embryo transfer cycles from January 2018 to December 2020 were involved. Associations between glucose metabolism indicators, adiposity and lipid metabolism indicators, and IVF/ICSI outcomes were explored using multivariable generalized linear models. Mediation analyses were further performed to examine the potential mediation role of adiposity and lipid metabolism indicators. RESULTS: Significant dose-dependent relationships were found between glucose metabolism indicators and IVF/ICSI early reproductive outcomes and between glucose metabolism indicators and adiposity and lipid metabolism indicators (all P < 0.05). Also, we found significant dose-dependent relationships between adiposity and lipid metabolism indicators and IVF/ICSI early reproductive outcomes (all P < 0.05). The mediation analysis indicated that elevated FPG, 2hPG, FPI, 2hPI, HbA1c, and HOMA2-IR were significantly associated with decreased retrieved oocyte count, MII oocyte count, normally fertilized zygote count, normally cleaved embryo count, high-quality embryo count, or blastocyst formation count after controlling for adiposity and lipid metabolism indicators. Serum TG mediated 6.0-31.0% of the associations; serum TC mediated 6.1-10.8% of the associations; serum HDL-C mediated 9.4-43.6% of the associations; serum LDL-C mediated 4.2-18.2% of the associations; and BMI mediated 26.7-97.7% of the associations. CONCLUSIONS: Adiposity and lipid metabolism indicators (i.e., serum TG, serum TC, serum HDL-C, serum LDL-C, and BMI) are significant mediators of the effect of glucose metabolism indicators on IVF/ICSI early reproductive outcomes in PCOS women, indicating the importance of preconception glucose and lipid management and the dynamic equilibrium of glucose and lipid metabolism in PCOS women.

Preconception genome medicine: current state and future perspectives to improve infertility diagnosis and reproductive and health outcomes based on individual genomic data.

BACKGROUND: Our genetic code is now readable, writable and hackable. The recent escalation of genome-wide sequencing (GS) applications in population diagnostics will not only enable the assessment of risks of transmitting well-defined monogenic disorders at preconceptional stages (i.e. carrier screening), but also facilitate identification of multifactorial genetic predispositions to sub-lethal pathologies, including those affecting reproductive fitness. Through GS, the acquisition and curation of reproductive-related findings will warrant the expansion of genetic assessment to new areas of genomic prediction of reproductive phenotypes, pharmacogenomics and molecular embryology, further boosting our knowledge and therapeutic tools for treating infertility and improving women's health. OBJECTIVE AND RATIONALE: In this article, we review current knowledge and potential development of preconception genome analysis aimed at detecting reproductive and individual health risks (recessive genetic disease and medically actionable secondary findings) as well as anticipating specific reproductive outcomes, particularly in the context of IVF. The extension of reproductive genetic risk assessment to the general population and IVF couples will lead to the identification of couples who carry recessive mutations, as well as sub-lethal conditions prior to conception. This approach will provide increased reproductive autonomy to couples, particularly in those cases where preimplantation genetic testing is an available option to avoid the transmission of undesirable conditions. In addition, GS on prospective infertility patients will enable genome-wide association studies specific for infertility phenotypes such as predisposition to premature ovarian failure, increased risk of aneuploidies, complete oocyte immaturity or blastocyst development failure, thus empowering the development of true reproductive precision medicine. SEARCH METHODS: Searches of the literature on PubMed Central included combinations of the following MeSH terms: human, genetics, genomics, variants, male, female, fertility, next generation sequencing, genome exome sequencing, expanded carrier screening, secondary findings, pharmacogenomics, controlled ovarian stimulation, preconception, genetics, genome-wide association studies, GWAS. OUTCOMES: Through PubMed Central queries, we identified a total of 1409 articles. The full list of articles was assessed for date of publication, limiting the search to studies published within the last 15 years (2004 onwards due to escalating research output of next-generation sequencing studies from that date). The remaining articles' titles were assessed for pertinence to the topic, leaving a total of 644 articles. The use of preconception GS has the potential to identify inheritable genetic conditions concealed in the genome of around 4% of couples looking to conceive. Genomic information during reproductive age will also be useful to anticipate late-onset medically actionable conditions with strong genetic background in around 2-4% of all individuals. Genetic variants correlated with differential response to pharmaceutical treatment in IVF, and clear genotype-phenotype associations are found for aberrant sperm types, oocyte maturation, fertilization or pre- and post-implantation embryonic development. All currently known capabilities of GS at the preconception stage are reviewed along with persisting and forthcoming barriers for the implementation of precise reproductive medicine. WIDER IMPLICATIONS: The expansion of sequencing analysis to additional monogenic and polygenic traits may enable the development of cost-effective preconception tests capable of identifying underlying genetic causes of infertility, which have been defined as 'unexplained' until now, thus leading to the development of a true personalized genomic medicine framework in reproductive health.

The effect of intralipid on pregnancy outcomes in women with previous implantation failure in in vitro fertilization/intracytoplasmic sperm injection cycles: A systematic review and meta-analysis.

Several recent studies have investigated the relationship between intravenous intralipid and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes in women with previous implantation failure. We conducted a systematic review and meta-analysis to evaluate the effects of intravenous intralipid on pregnancy outcomes in women with previous implantation failure. Ovid MEDLINE, the Cochrane Library, Embase and ClinicalTrials.gov were searched up to August 5th, 2019. Randomized controlled trials comparing intravenous intralipid with placebo or no treatment during IVF/ICSI cycles in women with at least one implantation failure were included. Results were presented as risk ratio (RR) with 95 % confidence intervals (CIs). Four studies with 544 participants were included. Live birth rate was statistically higher among the groups of women who received intravenous intralipid (RR 1.98, 95 % CI 1.39-2.80, quality of evidence: low). Intralipid infusion could significantly improve clinical pregnancy rate (RR 1.74, 95 % CI 1.27-2.40, quality of evidence: low). When excluding two studies only published as conference abstracts, there were no significant differences in terms of live birth (RR 1.78, 95 % CI 0.95-3.34, heterogeneity: I²â€¯= 25.5 %, quality of evidence: low, Fig. 4A) and clinical pregnancy (RR 1.66, 95 % CI 0.90-3.08, heterogeneity: I²â€¯= 47.7 %, quality of evidence: low, Fig. 4B). Adverse events were reported to be rare, but three congenital anomalies were observed in women receiving intravenous intralipid. Administering intravenous intralipid during IVF/ICSI cycles may improve live birth and clinical pregnancy in women with previous implantation failure, such benefit is not significant excluding studies with high risk of bias in the analysis, more studies are needed to evaluate its efficacy and safety especially congenital malformations.

Effects of chromosome 9 inversion on IVF/ICSI: A 7-year retrospective cohort study.

BACKGROUND: This study focused on the outcomes of patients with pericentric inversion of chromosome 9 who underwent IVF/ICSI and fresh day 2 or day 3 embryo transfer and the possible impacts of carrier gender and chromosome karyotype on pregnancy outcomes. METHODS: A total of 214 couples (107 couples with one pericentric inversion of chromosome 9 in one partner [Group 1], 107 couples with normal karyotypes [Group 2]) underwent their first IVF/ICSI treatment and were included in this study. Oocyte number, normal fertilization rates, abnormal fertilization rates, cleavage rates, embryo utilization rates, fresh embryo transfer rates, clinical pregnancy rates (CPR), implantation rates, miscarriage rates, and live birth rates per embryo transfer (LBR) were compared between groups. RESULTS: Group 1 did not show any disadvantage when compared with Group 2. The CPR and LBR were similar between all groups. The female carrier group had a higher normal fertilization rate and higher utilization rate than the male carrier group. Cases with inv(9)(p12;q13) had a lower utilization rate but a higher implantation rate than the remaining karyotypes. CONCLUSION: In the first IVF or ICSI cycle, couples with one pericentric inversion of chromosome 9 in one partner had satisfactory outcomes. The subgroup analysis showed a tendency of better prognosis for the female carrier and inv(9)(p12;q13) type.

Serum anti-Müllerian hormone is associated with oocyte dysmorphisms and ICSI outcomes.

OBJECTIVE: To evaluate the association between serum levels of anti-Müllerian hormone (AMH) and oocyte dysmorphisms in intracytoplasmic sperm injection (ICSI) cycles. METHODS: A retrospective study of data from 628 ICSI cycles with successful oocyte retrieval carried out at a single center in Tehran from November 2015 to July 2018. Cycles were divided into six groups by serum AMH level. Various oocyte dysmorphisms, quantity of retrieved oocytes, fertilization rates, cleavage-stage embryos, and pregnancy rates were compared among the groups. RESULTS: Serum AMH was associated with cytoplasm granulation, abnormally amorphous oocytes (P˂0.01), extended perivitelline space (P˂0.001), granulated perivitelline space (P˂0.05), fragmented polar body (P˂0.001), and average of oocyte quality index (AOQI) (P˂0.01). The total number of aspirated and metaphase ΙΙ oocytes increased with increasing AMH levels (P<0.001). There was no difference in the rate of fertilization or cleavage-stage embryos among the study groups; however, the pregnancy rate differed significantly (P<0.05). CONCLUSIONS: Serum levels of AMH were associated with specific oocyte dysmorphisms and AOQI. Serum AMH levels might influence both qualitative and quantitative aspects of the ovarian response to stimulation and also the pregnancy rate in ICSI cycles.

Predicting the clinical outcome of ICSI by sperm head vacuole examination.

To assess whether high magnification sperm head vacuole examination (SHVE) and/or standard sperm morphology assessment can predict ICSI outcomes in terms of fertilization, embryo quality, and delivery rates, a prospective observational bicentric study was conducted in two publicly funded assisted reproductive technology (ART) units in France between January and July of 2012. A total of 111 ICSI cycles for exclusively male infertility factors were included. A Spearman's correlation test was performed to validate SHVE reproducibility between the ART units. The normal morphology rate and SHVE performed on selected spermatozoa were respectively determined according to David's and Vanderzwalmen's classifications used for motile sperm organelle morphology examination (MSOME) on the day of the ICSI. Receiver Operating Characteristic (ROC) curve analysis was performed to determine thresholds associated with the occurrence of a delivery. There was an excellent correlation between the two operators (r=0.98), thus validating the study's SHVE data. Percentages of normal morphology grade spermatozoa using the standard classification and first-best morphology grade spermatozoa determined by SHVE were not significantly associated with (i) delivery (p=0.58; 0.90 /area under curve (AUC) =0.532; 0.507), (ii) fertilization (p=0.88; 0.90), (iii) top-quality embryos (p=0.27; 0.98), and (iv) good quality embryo rates (p=0.73; 0.98), respectively. In conclusion, high magnification SHVE and standard sperm morphology assessment cannot predict clinical or biological ICSI outcomes. ABBREVIATIONS: ART: assisted reproductive technology; HBV: hepatitis B virus; HCV: hepatitis C virus; HIV: human immunodeficiency virus; ICSI: intra-cytoplasmic sperm injection; IVF: in vitro fertilization; LNVs: large nuclear vacuoles; MSOME: motile sperm organelle morphology examination; SHVE: sperm head vacuole examination; WHO: World Health Organization.