密度梯度離心 (DGC)  vs DGC 後進行磁活化細胞分選 (MACS) vs 使用精子分離裝置 (SSD)

傳統 DGC 相比，MACS 和 SSD 後的 DFI (DNA fragmentation index)

均顯著降低

SSD 和 MACS 之間的進一步比較表明，SSD 組中凋亡細胞明顯較少。

Observational Study

 

. 2024 Aug;41(8):2201-2209.

 doi: 10.1007/s10815-024-03168-9. Epub 2024 Jun 18.

Optimized sperm selection: a highly efficient device for the isolation of progressive motile sperm with low DNA fragmentation index

Purpose: To identify the sperm preparation procedure that selects the best sperm population for medically assisted reproduction.

Methods: Prospective observational study comparing the effect of four different sperm selection procedures on various semen parameters. Unused raw semen after routine diagnostic analysis was split in four fractions and processed by four different methods: (1) density gradient centrifugation (DGC), (2) sperm wash (SW), (3) DGC followed by magnetic activated cell sorting (MACS), and (4) using a sperm separation device (SSD). Each fraction was analyzed for progressive motility, morphology, acrosome index (AI), and DNA fragmentation index (DFI).

Results: With DGC as standard of care in intraclass correlation coefficient analysis, only SSD was in strong disagreement regarding progressive motility and DFI [0.26, 95%CI (- 0.2, 0.58), and 0.17, 95%CI (- 0.19, 0.45), respectively]. When controlling for abstinence duration, DFI was significantly lower after both MACS and SSD compared to DGC [- 0.27%, 95%CI (- 0.47, - 0.06), p = 0.01, and - 0.6%, 95%CI (- 0.80, - 0.41), p < 0.001, respectively]. Further comparisons between SSD and MACS indicate significantly less apoptotic cells [Median (IQR) 4 (5), 95%CI (4.1, - 6.8) vs Median (IQR) 5 (8), 95%CI (4.9, - 9.2), p < 0.001, respectively] and dead cells [Median (IQR) 9.5 (23.3), 95%CI (13.2, - 22.4) vs Median (IQR) 22 (28), 95%CI (23.1, - 36.8), p < 0.001, respectively] in the SSD group.

Conclusion: The selection of a population of highly motile spermatozoa with less damaged DNA from unprocessed semen is ideally performed with SSD. Question remains whether this method improves the embryological outcomes in the IVF laboratory.

接受微流控精子製備的患者與接受標準精子處理進行體外受精 (IVF) 和卵胞漿內單精子注射 (ICSI) 的患者相比，其卵裂期和囊胚期胚胎質量以及臨床妊娠率和持續妊娠率相似。

Randomized Controlled Trial

 

. 2022 Jun 30;37(7):1406-1413.

 doi: 10.1093/humrep/deac099.

Microfluidic preparation of spermatozoa for ICSI produces similar embryo quality to density-gradient centrifugation: a pragmatic, randomized controlled trial

Study question: Does processing of spermatozoa for IVF with ICSI by a microfluidic sperm separation device improve embryo quality compared with density-gradient centrifugation?

Summary answer: Patients randomized to microfluidic sperm preparation had similar cleavage- and blastocyst-stage embryo quality and clinical and ongoing pregnancy rates to those who underwent standard sperm processing for IVF with ICSI.

What is known already: Microfluidic sperm preparation can isolate spermatozoa for clinical use with minimal DNA fragmentation but with unclear impact on clinical outcomes.

Study design, size, duration: A prospective randomized controlled trial of 386 patients planning IVF from June 2017 through September 2021 was carried out.

Participants/materials, setting, methods: One hundred and ninety-two patients were allocated to sperm processing with a microfluidic sperm separation device for ICSI, while 194 patients were allocated to clinical standard density-gradient centrifugation (control) at an academic medical centre.

Main results and the role of chance: In an intention to treat analysis, there were no differences in high-quality cleavage-stage embryo fraction [66.0 (25.8)% control versus 68.0 (30.3) microfluidic sperm preparation, P = 0.541, absolute difference -2.0, 95% CI (-8.5, 4.5)], or high-quality blastocyst fraction [37.4 (25.4) control versus 37.4 (26.2) microfluidic sperm preparation, P = 0.985, absolute difference -0.6 95% CI (-6, 5.9)] between groups. There were no differences in the clinical pregnancy or ongoing pregnancy rates between groups.

吸菸、高齡和環境污染物  環境因會影響精子DNA甲基化組。

男性生育力低下和精液參數異常，尤其是少精子症，似乎與精子DNA甲基化異常有關。

目前尚無確鑿證據顯示精子DNA甲基化的變化會影響懷孕後代健康。

. 2020 Nov 1;26(6):841-873.

 doi: 10.1093/humupd/dmaa025.

DNA methylation in human sperm: a systematic review

Background: Studies in non-human mammals suggest that environmental factors can influence spermatozoal DNA methylation, and some research suggests that spermatozoal DNA methylation is also implicated in conditions such as subfertility and imprinting disorders in the offspring. Together with an increased availability of cost-effective methods of interrogating DNA methylation, this premise has led to an increasing number of studies investigating the DNA methylation landscape of human spermatozoa. However, how the human spermatozoal DNA methylome is influenced by environmental factors is still unclear, as is the role of human spermatozoal DNA methylation in subfertility and in influencing offspring health.

Objective and rationale: The aim of this systematic review was to critically appraise the quality of the current body of literature on DNA methylation in human spermatozoa, summarize current knowledge and generate recommendations for future research.

Search methods: A comprehensive literature search of the PubMed, Web of Science and Cochrane Library databases was conducted using the search terms 'semen' OR 'sperm' AND 'DNA methylation'. Publications from 1 January 2003 to 2 March 2020 that studied human sperm and were written in English were included. Studies that used sperm DNA methylation to develop methodologies or forensically identify semen were excluded, as were reviews, commentaries, meta-analyses or editorial texts. The Grading of Recommendations, Assessment, Development and Evaluations (GRADE) criteria were used to objectively evaluate quality of evidence in each included publication.

Outcomes: The search identified 446 records, of which 135 were included in the systematic review. These 135 studies were divided into three groups according to area of research; 56 studies investigated the influence of spermatozoal DNA methylation on male fertility and abnormal semen parameters, 20 studies investigated spermatozoal DNA methylation in pregnancy outcomes including offspring health and 59 studies assessed the influence of environmental factors on spermatozoal DNA methylation. Findings from studies that scored as 'high' and 'moderate' quality of evidence according to GRADE criteria were summarized. We found that male subfertility and abnormal semen parameters, in particular oligozoospermia, appear to be associated with abnormal spermatozoal DNA methylation of imprinted regions. However, no specific DNA methylation signature of either subfertility or abnormal semen parameters has been convincingly replicated in genome-scale, unbiased analyses. Furthermore, although findings require independent replication, current evidence suggests that the spermatozoal DNA methylome is influenced by cigarette smoking, advanced age and environmental pollutants. Importantly however, from a clinical point of view, there is no convincing evidence that changes in spermatozoal DNA methylation influence pregnancy outcomes or offspring health.

 嚴重少精症會造成ICSI後

受孕率下降

延長胚胎第2次分裂時間

D3胚胎品質

The effect of severe oligozoospermia on embryo dynamics and morphological quality

Purpose

This study evaluated whether severe oligozoospermia affects early cleavage-stage dynamics and blastocyst developmental competence in ICSI cycles cultured in a time-lapse system.

Methods

This retrospective matched cohort study included ICSI cycles using fresh ejaculated sperm and fresh autologous oocytes at Assisted Reproductive Center, Tam Anh Hanoi General Hospital (February 2022–December 2024). Initially, eligible cycles were identified in both groups. After propensity score matching (1:2), the final analytic cohort comprised 39 cycles in the severe oligozoospermia group and 78 cycles in the normozoospermia group. All normally fertilized zygotes (2PN) were monitored up to day 7. Cleavage-stage morphokinetic parameters and abnormal cleavage patterns were analyzed. Multivariate analyses evaluated overall morphokinetic differences. A generalized linear mixed model (GLMM) tested predictors of usable blastocysts, comparing 4095 candidate models based on the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC).

Results

A total of 117 ICSI cycles were analyzed after propensity score matching, including 39 cycles with severe oligozoospermia and 78 normozoospermic controls. Despite a significantly higher number of retrieved MII oocytes, the severe oligozoospermia group exhibited lower fertilization rates (71.33% vs. 77.98%, p = 0.012) and a reduced proportion of top-quality day-3 embryos (51.25% vs. 65.82%, p < 0.001). Blastocyst formation and top-quality blastocyst rates did not differ significantly between groups. Cleavage-stage morphokinetic analysis revealed a statistically significant prolongation of the second cell cycle (ECC2) in the severe oligozoospermia group; however, the absolute difference was small (~ 0.25 h) and accounted for a limited proportion of variance (R² ≈ 1.4%). No significant differences were observed in subsequent morphokinetic parameters, including ECC3 or the synchrony parameters s2 and s3. In generalized linear mixed-model analysis using embryo usability (transfer or cryopreservation) as a laboratory-defined surrogate outcome, sperm concentration was not identified as an independent predictor.

Conclusions

Severe oligozoospermia was associated with impaired fertilization outcomes and reduced day-3 embryo quality. A modest but statistically significant prolongation of ECC2 was observed; however, when sperm concentration was evaluated in isolation under ICSI conditions, later cleavage-stage morphokinetics and blastocyst development appeared largely preserved at the laboratory level. These findings are limited to sperm concentration alone and may be influenced by unmeasured sperm functional defects.

 10種以上gene  影響精蟲頸部的蛋白質 中心粒及胞器  其基因突變會造成精蟲斷頭現象(從精蟲頸部斷裂)

Review

 

. 2026 Feb;43(2):347-365.

 doi: 10.1007/s10815-025-03769-y. Epub 2026 Jan 5.

The spermatozoon neck role in infertility and intracytoplasmic sperm injection outcomes

The mammalian spermatozoon neck is a unique structure that functions during spermatid differentiation and spermatozoa swimming, and its contents are critical for post-fertilization embryogenesis. Mutations in proteins localizing to the neck connecting piece (the modified pericentriolar material) result in acephalic spermatozoa. In contrast, mutations in proteins localizing to the centriole often produce abnormal tail morphology. Acephalic spermatozoa can be categorized based on the exact location of the neck breakpoint. Here, we classify 24 proteins known to cause acephaly in human and mice spermatozoa into five different acephalic types, depending on where the breakpoint occurs. We also discuss other proteins found in the spermatozoon neck, which may result in spermatozoa acephaly. The relationship between the exact location of the neck's break and intracytoplasmic sperm injection (ICSI) outcomes is explored in the context of the spermatozoon centrosome's role. We conclude that to understand this relationship, future research should investigate DNA, phospholipase C zeta, and centriole functionality, in addition to the location of the acephalic breakpoint in the patient's sperm.

 31 個基因的突變會導致頭部缺陷，62 個基因的突變會導致精子尾部缺陷。

基因突變導致精子形態畸形/畸形精子症

Review

 

. 2024 Nov;41(11):2877-2929.

 

Genetic etiological spectrum of sperm morphological abnormalities

Purpose: Male infertility manifests in the form of a reduction in sperm count, sperm motility, or the loss of fertilizing ability. While the loss of sperm production can have mixed reasons, sperm structural defects, cumulatively known as teratozoospermia, have predominantly genetic bases. The aim of the present review is to undertake a comprehensive analysis of the genetic mutations leading to sperm morphological deformities/teratozoospermia.

Methods: We undertook literature review for genes involved in sperm morphological abnormalities. The genes were classified according to the type of sperm defects they cause and on the basis of the level of evidence determined by the number of human studies and the availability of a mouse knockout.

Results: Mutations in the SUN5, CEP112, BRDT, DNAH6, PMFBP1, TSGA10, and SPATA20 genes result in acephalic sperm; mutations in the DPY19L2, SPATA16, PICK1, CCNB3, CHPT1, PIWIL4, and TDRD9 genes cause globozoospermia; mutations in the AURKC gene cause macrozoospermia; mutations in the WDR12 gene cause tapered sperm head; mutations in the RNF220 and ADCY10 genes result in small sperm head; mutations in the AMZ2 gene lead to vacuolated head formation; mutations in the CC2D1B and KIAA1210 genes lead to pyriform head formation; mutations in the SEPT14, ZPBP1, FBXO43, ZCWPW1, KATNAL2, PNLDC1, and CCIN genes cause amorphous head; mutations in the SEPT12, RBMX, and ACTL7A genes cause deformed acrosome formation; mutations in the DNAH1, DNAH2, DNAH6, DNAH17, FSIP2, CFAP43, AK7, CHAP251, CFAP65, ARMC2 and several other genes result in multiple morphological abnormalities of sperm flagella (MMAF).

Conclusions: Altogether, mutations in 31 genes have been reported to cause head defects and mutations in 62 genes are known to cause sperm tail defects.

IVF或ICSI前，將收集的卵子在特製培養基中培養3-6H，可提高卵子的成熟度和早期胚胎發育品質。

Review

 

. 2025 May 2;24(1):e12652.

 doi: 10.1002/rmb2.12652. eCollection 2025 Jan-Dec.

The quality of human eggs and its pre-IVF incubation

Background: Multi-factors influence the success rate of infertility treatments, and one of the important points is to obtain good quality eggs. Methods: Based on the literatures and unpublished data, the factors affecting egg quality were summarized.

Main findings results: Egg quality is an important determinant in successful infertility treatment. In addition to maternal age, controlled ovarian hyperstimulation (COH) protocols also play a key role in affecting the quality of the egg. After egg retrieval, the insemination occurs 3-6 h after collection, with a pre-IVF incubation time by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (39-42 h post-HCG injection). The pre-IVF incubation refers to the short period time of 3 to 6 h after oocyte retrieval and before the insemination by IVF or ICSI. The pre-IVF incubation of collected eggs in the designed culture medium improves egg quality in terms of maturation and early embryonic development.

Conclusions: Pre-IVF incubation of the collected eggs contributes to the improvement of the quality of eggs; therefore, it may increase subsequent pregnancy and implantation rates following embryo transfer.

鮮胚植入或凍胚植入在懷孕率方面並沒有明顯差異

兩者在累積懷孕率方面也沒有明顯差異

凍胚植入的最大好處是可以降低卵巢過度刺激的機率OHSS

Meta-Analysis

 

. 2021 Feb 4;2(2):CD011184.

 doi: 10.1002/14651858.CD011184.pub3.

Fresh versus frozen embryo transfers in assisted reproduction

Background: In vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatments conventionally consist of a fresh embryo transfer, possibly followed by one or more cryopreserved embryo transfers in subsequent cycles. An alternative option is to freeze all suitable embryos and transfer cryopreserved embryos in subsequent cycles only, which is known as the 'freeze all' strategy. This is the first update of the Cochrane Review on this comparison. Objectives: To evaluate the effectiveness and safety of the freeze all strategy compared to the conventional IVF/ICSI strategy in women undergoing assisted reproductive technology. Search methods: We searched the Cochrane Gynaecology and Fertility Group Trials Register, CENTRAL, MEDLINE, Embase, PsycINFO, CINAHL, and two registers of ongoing trials from inception until 23 September 2020 for relevant studies, checked references of publications found, and contacted study authors to obtain additional data. Selection criteria: Two review authors (TZ and MZ) independently selected studies for inclusion, assessed risk of bias, and extracted study data. We included randomised controlled trials comparing a 'freeze all' strategy with a conventional IVF/ICSI strategy including a fresh embryo transfer in women undergoing IVF or ICSI treatment. Data collection and analysis: The primary outcomes were cumulative live birth rate and ovarian hyperstimulation syndrome (OHSS). Secondary outcomes included effectiveness outcomes (including ongoing pregnancy rate and clinical pregnancy rate), time to pregnancy and obstetric, perinatal and neonatal outcomes. Main results: We included 15 studies in the systematic review and eight studies with a total of 4712 women in the meta-analysis. The overall evidence was of moderate to low quality. We graded all the outcomes and downgraded due to serious risk of bias, serious imprecision and serious unexplained heterogeneity. Risk of bias was associated with unclear blinding of investigators for preliminary outcomes of the study during the interim analysis, unit of analysis error, and absence of adequate study termination rules. There was an absence of high-quality evidence according to GRADE assessments for our primary outcomes, which is reflected in the cautious language below. There is probably little or no difference in cumulative live birth rate between the 'freeze all' strategy and the conventional IVF/ICSI strategy (odds ratio (OR) 1.08, 95% CI 0.95 to 1.22; I2 = 0%; 8 RCTs, 4712 women; moderate-quality evidence). This suggests that for a cumulative live birth rate of 58% following the conventional strategy, the cumulative live birth rate following the 'freeze all' strategy would be between 57% and 63%. Women might develop less OHSS after the 'freeze all' strategy compared to the conventional IVF/ICSI strategy (OR 0.26, 95% CI 0.17 to 0.39; I2 = 0%; 6 RCTs, 4478 women; low-quality evidence). These data suggest that for an OHSS rate of 3% following the conventional strategy, the rate following the 'freeze all' strategy would be 1%. There is probably little or no difference between the two strategies in the cumulative ongoing pregnancy rate (OR 0.95, 95% CI 0.75 to 1.19; I2 = 31%; 4 RCTs, 1245 women; moderate-quality evidence). We could not analyse time to pregnancy; by design, time to pregnancy is shorter in the conventional strategy than in the 'freeze all' strategy when the cumulative live birth rate is comparable, as embryo transfer is delayed in a 'freeze all' strategy. We are uncertain whether the two strategies differ in cumulative miscarriage rate because the evidence is very low quality (Peto OR 1.06, 95% CI 0.72 to 1.55; I2 = 55%; 2 RCTs, 986 women; very low-quality evidence) and cumulative multiple-pregnancy rate (Peto OR 0.88, 95% CI 0.61 to 1.25; I2 = 63%; 2 RCTs, 986 women; very low-quality evidence). The risk of hypertensive disorders of pregnancy (Peto OR 2.15, 95% CI 1.42 to 3.25; I2 = 29%; 3 RCTs, 3940 women; low-quality evidence), having a large-for-gestational-age baby (Peto OR 1.96, 95% CI 1.51 to 2.55; I2 = 0%; 3 RCTs, 3940 women; low-quality evidence) and a higher birth weight of the children born (mean difference (MD) 127 g, 95% CI 77.1 to 177.8; I2 = 0%; 5 RCTs, 1607 singletons; moderate-quality evidence) may be increased following the 'freeze all' strategy. We are uncertain whether the two strategies differ in the risk of having a small-for-gestational-age baby because the evidence is low quality (Peto OR 0.82, 95% CI 0.65 to 1.05; I2 = 64%; 3 RCTs, 3940 women; low-quality evidence). Authors' conclusions: We found moderate-quality evidence showing that one strategy is probably not superior to the other in terms of cumulative live birth rate and ongoing pregnancy rate. The risk of OHSS may be decreased in the 'freeze all' strategy. Based on the results of the included studies, we could not analyse time to pregnancy. It is likely to be shorter using a conventional IVF/ICSI strategy with fresh embryo transfer in the case of similar cumulative live birth rate, as embryo transfer is delayed in a 'freeze all' strategy. The risk of maternal hypertensive disorders of pregnancy, of having a large-for-gestational-age baby and a higher birth weight of the children born may be increased following the 'freeze all' strategy. We are uncertain if 'freeze all' strategy reduces the risk of miscarriage, multiple pregnancy rate or having a small-for-gestational-age baby compared to conventional IVF/ICSI.

培養基系統葡萄糖濃度呈現高-低-高的模式：受精培養基為 2.5-3 mM，卵裂期培養基為 0.5 mM 或更低，囊胚期培養基為 2.5-3.3 mM。

. 2025 Jan 1;40(1):30-40.

 doi: 10.1093/humrep/deae248.

The composition of commercially available human embryo culture media

Study question: What is the composition of currently available commercial human embryo culture media provided by seven suppliers, for each stage of human preimplantation embryo development?

Summary answer: While common trends existed across brands, distinct differences in composition underlined the absence of a clear standard for human embryo culture medium formulation.

What is known already: The reluctance of manufacturers to fully disclose the composition of their human embryo culture media generates uncertainty regarding the culture conditions that are used for human preimplantation embryo culture. The critical role of the embryo culture environment is well-recognized, with proven effects on IVF success rates and child outcomes, such as birth weight. The lack of comprehensive composition details restricts research efforts crucial for enhancing our understanding of its impacts on these outcomes. The ongoing demand for greater transparency remains unmet, highlighting a significant barrier in embryo culture medium optimization.

Study design, size, duration: For this study, 47 different human embryo culture media and protein supplements were purchased between December 2019 and June 2020; they comprise complete media (n = 23), unsupplemented media (n = 14), and supplements (n = 10). Unsupplemented media were supplemented with each available supplement from the same brand (n = 33 combinations). All samples were directly frozen in liquid nitrogen and stored at -80°C until composition analysis.

Participants/materials, setting, methods: We determined the concentrations of 40 components in all samples collected (n = 80). Seven electrolytes (calcium, chloride, iron, magnesium, phosphate, potassium, sodium), glucose, immunoglobulins A, G, and M (IgA, IgG, IgM), uric acid, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and albumin, as well as the total protein concentration, were determined in each sample using a Cobas 8000 Analyser (Roche Diagnostics). Analysis of pyruvate, lactate, carnitine, and 21 amino acids was achieved with Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS).

Main results and the role of chance: Our analysis showed that generally, the concentrations of components of ready-to-use human embryo culture media align with established assumptions about the changing needs of an embryo during early development. For instance, glucose concentrations displayed a high-low-high pattern in sequential media systems from all brands: 2.5-3 mM in most fertilization media, 0.5 mM or below in all cleavage stage media, and 2.5-3.3 mM in most blastocyst stage media. Continuous media generally resembled glucose concentrations of cleavage stage media. However, for other components, such as lactate, glycine, and potassium, we observed clear differences in medium composition across different brands. No two embryo culture media compositions were the same. Remarkably, even embryo culture media from brands that belong to the same parent company differed in composition. Additionally, the scientific backing for the specific concentrations used and the differences in the composition of sequential media is quite limited and often based on minimal in vivo studies of limited sample size or studies using animal models.

三個正常染色體囊胚可提供93%的生育數率。

冷凍卵子時年齡小於35歲，平均需15個成熟卵細胞才能獲得三個正常染色體囊胚。

若患者年齡≥38歲，所成熟卵細胞數量增加一倍；，平均需30個成熟卵細胞才能獲得三個正常染色體囊胚。

若患者年齡≥40歲，則所需MII期卵母細胞數量增加至三倍，，平均需45個成熟卵細胞才能獲得三個正常染色體囊胚。。 

應鼓勵38歲之前進行卵子冷凍。

年齡≥38歲的女性，可能需要多次冷凍卵子週期才能獲得較高的活產率。

. 2025 Sep;124(3):487-495.

 doi: 10.1016/j.fertnstert.2025.04.023. Epub 2025 Apr 23.

The number of autologous, vitrified mature oocytes needed to obtain three euploid blastocysts increases with age

Objective: To determine the number of mature oocytes required to make at least three euploid blastocysts.

Subjects: All patients at a multicenter assisted reproductive technology practice that warmed vitrified autologous oocytes for in vitro fertilization from 2011 to October 2023.

Main outcome measures: Number of mature (metaphase II [MII]) oocytes to achieve at least three euploid blastocysts.

Results: A total of 1,041 thaw cycles from 986 patients were analyzed. Patients were stratified by age at vitrification. Measures of ovarian reserve decreased with advancing age, as expected. The number of mature oocytes (MII) banked and euploid blastocysts available decreased as age at vitrification increased. Patients aged 35-40 years used preimplantation genetic testing more than those older than 40 years or younger than 35 years. To optimize the chance of a potential live birth by banking at least three euploid blastocysts, patients aged <35 years at vitrification needed a mean of 15 MII oocytes. This number doubled in patients aged ≥38 years and tripled for patients aged >40 years. We also analyzed potential live birth per number of MII oocytes banked, which also increased with age, with 0.13 expected live births per thawed MII oocyte at the age of <35 years and 0.04 expected live births if older than 40 years at time of oocyte vitrification. Age at vitrification was more influential than indication for vitrification for expected outcome of live birth.

Conclusion: From previously published literature, three euploid blastocysts provided a 93% chance of a child. From our analysis, patients required a mean of 15 mature oocytes to yield at least three euploid blastocysts if they were aged <35 years at time of vitrification. The required number of MII oocytes doubled in patients aged ≥38 years and tripled in those aged ≥40 years, to achieve the same goal. Given our results, we should encourage egg freeze patients to cryopreserve before the age of 38 years. We should also counsel patients that multiple vitrification cycles will likely be required to achieve a high probability of live birth from future oocyte thaws for women aged ≥38 years.