Developmental potential of immature human oocytes aspirated after controlled ovarian stimulation

Purpose

Intracytoplasmic sperm injection (ICSI) for initially immature oocytes that mature in vitro is controversial and practice varies widely. While it may increase the number of usable embryos, it may also be time-intensive and potentially low-yield. This study sought to elucidate which patients may benefit from ICSI of initially immature oocytes that matured in vitro.

Methods

A retrospective study comparing fertilization, cleavage, blastulation, and embryo usage rates between sibling initially immature and mature oocytes that underwent ICSI between 2015 and 2019 was performed. Outcomes of initially immature oocytes were stratified by initial maturation stage, timing of progression to metaphase II (MII) in vitro, percentage of mature oocytes in the cycle, and female age.

Results

Ten thousand eight hundred seventeen oocytes from 889 cycles were included. Of 3137 (29.0%) initially immature oocytes, 418 (13.3%) reached MII later on the day of retrieval (day 0) and 1493 (47.6%) on day 1. Overall, embryos originating from initially immature oocytes had lower cleavage and blastulation rates compared to those from initially mature oocytes (P<0.05, all groups). However, embryos from oocytes that matured later on day 0 comprised a unique subset that had clinically similar cleavage (75% vs 80%, RR 0.93, P=0.047) and blastulation rates (41% vs 50%, RR 0.81, P=0.024) compared to initially mature oocytes. Women with low percentages of mature oocytes in the cycle overall and women ≥40 in cleavage cycles derived the highest relative benefit from the use of immature oocytes.

Conclusion

ICSI of immature oocytes, particularly those that mature later on the day of retrieval, may improve numbers of usable embryos. This study supports routine reassessment of immature oocytes for progression to MII and ICSI on day 0. An additional reassessment on day 1 may also be of use in older women or those with low percentage of mature oocytes.

     

A randomized,non-inferiority trial on the DuoStim strategy in PGT-A cycles

Research questionIs the DuoStim strategy an effective alternative to two conventional ovarian stimulation cycles in poor-prognosis patients undergoing preimplantation genetic testing for aneuploidies (PGT-A) to improve euploidy rates and obtain the first euploid embryo in less time?DesignThis randomized controlled trial was performed at IVI Madrid between June 2017 and December 2020 and included 80 patients with a suboptimal profile aged 38 or older undergoing PGT-A cycles. Patients were blindly randomized into two groups: 39 women underwent two ovarian stimulations in consecutive cycles (control group), whereas the double stimulation strategy was applied to 41 women (DuoStim group). The main outcome was the euploidy rate in each group. The secondary outcomes were the time it took to obtain a euploid embryo and the main cycle outcomes.ResultsThe baseline characteristics of the patients were similar. No differences were found between the control group and the DuoStim group in the mean days of stimulation (21.3 ± 1.6 versus 23.0 ± 1.4, P = 0.10), total gonadotrophins (4005 ± 450 versus 4245 ± 430, P = 0.43), metaphase II oocytes (8.7 ± 1.8 versus 6.8 ± 1.7, P = 0.15) or euploid embryos obtained (0.8 ± 0.4 versus 0.6 ± 0.4, P = 0.45). The euploid rate per randomized patient (ITT) was 16.1% in the control group versus 22.7% in the DuoStim group, with P-values of 0.371, and the euploidy rate per patient treated was 39.0% versus 45.7% in the control versus DuoStim groups. However, there was a significant difference in the average number of days it took to obtain a euploid blastocyst, favouring the DuoStim group (44.1 ± 2.0 versus 23.3 ± 2.8, P < 0.001).ConclusionsThe use of the DuoStim strategy in poor-prognosis patients undergoing PGT-A cycles maintains a similar euploidy rate while reducing the time required to obtain a euploid blastocyst.     

The effect of vitrification on maturation and viability capacities of immature human oocytes

Background

15 % of oocytes collected from Assisted Reproductive Technology (ART) cycles are immature. These oocytes may be matured following in vitro maturation (IVM) program. It is possible to cryopreserve the immature oocytes for further use in ART after application of IVM.

Objective

The aim was to determine the maturation rate and viability of human oocytes that were matured in vitro after vitrification program.

Materials and methods

63 women (19–43 years old) who underwent controlled ovarian stimulation for ART were included in this study. 53 immature oocytes were used for fresh group (fIVM) and 50 immature oocytes for vitrification group (vIVM). The maturation medium was Ham’s F₁₀ supplemented with 0.75 IU FSH, 0.75 IU LH and 40 % human follicular fluid (HFF). After 36 h, maturation and morphology of all oocytes were assessed. Also, the oocyte viability was assessed using PI/Hoechst immunostaining technique.

Results

The maturation rates were reduced in vIVM group (56.0 %) in comparison to fIVM group (88.7 %; P < 0.001). Oocyte viability rate were also reduced in vIVM group (56.0 %) in comparison to fIVM (86.8 %, P < 0.007).

Conclusions

Cryopreservation via vitrification reduced both the maturation capacity and viability of human oocytes in IVM technology. It is, therefore, recommended to apply IVM on fresh immature oocytes, instead.     

Cryopreservation of immature and in-vitro matured human oocytes by vitrification

The objective of this study was to determine the efficacy of vitrification of human oocytes before and after in-vitro maturation (IVM). The immature oocytes recovered (n = 472) were divided into two groups: (i) immature oocytes (n = 219) vitrified at the germinal vesicle (GV) stage; and (ii) immature GV-stage oocytes (n = 253) that were firstly matured in vitro (MII-stage oocytes; n = 178), then vitrified (n = 79). The remaining oocytes (n = 99), which were not vitrified, were processed as controls. After warming, the oocyte survival, maturation and fertilization rates, as well as embryonic development, were compared. The results showed no significant difference between the survival rates of the oocytes vitrified at GV stage and those vitrified at MII stage (85.4% versus 86.1%). However, oocyte maturation rates were significantly reduced (P < 0.05) when oocytes were vitrified at immature GV stage followed by IVM (50.8%) in comparison with the control group (70.4%). Following insemination by intracytoplasmic sperm injection, there was no difference in the fertilization (62.1% versus 58.8%), cleavage (69.5% versus 67.5%) and blastocyst development (0.0% versus 0.0%) rates between these two groups. However, these results were significantly lower (P < 0.05) than those achieved in the control group. This suggests that better results can be achieved by vitrifying mature oocytes rather than immature oocytes.     

Morphokinetic characteristics of embryos derived from in-vitro-matured oocytes and their in-vivo-matured siblings after ovarian stimulation

Research question

Does delayed maturation of aspirated metaphase I (MI) oocytes, completed in vitro, adversely affect early embryo development?

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.     

Fertilization, embryo development, and clinical outcome of immature oocytes obtained from natural cycle in vitro fertilization

Purpose

To analyze the fertilization, embryo development, and clinical outcome of immature oocytes obtained from natural cycle IVF in women with regular cycles.

Methods

Natural cycle IVF was performed in 28 patients who had normal ovaries, > 6 antral follicle counts and were less than 40 years old (n = 28 cycles). An hCG trigger of 10,000 IU was administered 36 h before oocyte collection when the diameter of the dominant follicle (DF) was over 12 mm. Oocytes were retrieved from DF as well as from the cohort of smaller follicles. Embryological aspects of the mature and immature oocytes retrieved from these cycles as well as the implantation and clinical pregnancy rates depending on the origin of the embryos transferred were evaluated.

Result(s)

Overall clinical pregnancy and implantation rates were 20.8 % and 6.7 %, respectively. There were no differences in in vitro maturation (IVM), fertilization and embryo development between immature oocytes retrieved with and without in vivo matured oocytes. However, the clinical and implantation rates in cycles with embryos produced from in vivo matured oocytes transferred were better than the cycles where only IVM embryos were transferred (30.8 %, 9.1 % vs. 9.1 %, 3.2 %).

Conclusion(s)

Although our results show that immature oocytes from natural cycle IVF can fertilize normally and can be used to increase the number of embryos available for transfer, the embryos derived from the immature oocytes in natural cycles IVF have a poorer reproductive potential.     

Successful birth after transfer of blastocysts derived from oocytes of unstimulated woman with regular menstrual cycle after IVM approach

Purpose : To report a delivery after transfer of blastocysts derived from eggs collected following in vivo HCG priming in a patient with regular menstrual cycles undergoing in vitro maturation (IVM) program. Methods : A woman had regular menstrual cycle and had experience of ovarian hyperstimulation syndrome (OHSS) during a previous conventional IVF-ET cycle. The patient was primed with 10,000 IU HCG 36 h before egg retrieval. After oocyte collection, the maturity of oocytes was evaluated and immature oocytes were cultured in IVM medium. The matured oocytes were fertilized with husband sperm, and normal fertilized eggs were cultured to blastocysts stage until embryo transfer in uteri. Results : Three MII-stage and 13 GV-stage oocytes were collected from the patient. Three mature oocytes were fertilized by conventional IVF. All three fertilized oocytes were developed to blastocysts. Immature oocytes were matured in vitro and insemination was carried out by ICSI. Out of eight fertilized zygotes, two developed to blastocyst stage. Transfer of three expanded blastocysts on Day 6 resulted in pregnancy in the patient and one healthy baby was born. Conclusions : This report provides an approach to treat infertile women with regular menstrual cycle and high risk of OHSS.     

Outcome of gestational surrogacy according to IVF protocol

Purpose

Surrogacy remains the only option for having a biologic child for a unique population of women with severe medical conditions. However, no study has looked at surrogacy outcome as a result of the type of ovarian stimulation of the intended mother [controlled ovarian stimulation (COH), modified natural cycle (MNC), and in vitro maturation (IVM)] for oocyte retrieval.

Methods

This is a retrospective study, including all intended mothers and gestational carriers in a tertiary, university affiliated, medical center, from 1998 to 2016.

Results

Fifty-two women underwent 252 oocyte retrieval cycles. The pregnancy outcome of 212 embryo transfer cycles (64 gestational carriers) was reviewed according to the origin of the embryo. The number of retrieved oocytes was significantly higher following COH (n?=?132) compared with IVM (n?=?58) and MNC cycles (n?=?62) (p?=?0.013 and p?<?0.0001, respectively). Pregnancy rates for embryos transferred according to each protocol were similar. All pregnancies that ended in live births when oocytes from IVM cycles were used derived from transfers of retrieved mature and mixed mature and immature oocytes. Pregnancies that involved embryos derived solely from immature oocytes that further matured in vitro and were transferred to gestational carriers were unsuccessful.

Conclusions

MNC protocol is a good option to achieve pregnancy for intended mothers using gestational surrogacy who have contraindications to COH. The yield of IVM cycles in which immature oocytes are retrieved is inconclusive.

     

Dual stimulation using corifollitropin alfa in 54 Bologna criteria poor ovarian responders – a case series

Research question

What are the reproductive outcomes of Bologna criteria poor responders undergoing dual stimulation (DuoStim) and subsequent cryopreserved embryo transfer?

New alternative to infertility treatment for women without ovarian stimulation

Natural cycle IVF produced the world first successful live birth, but slowly this treatment has been replaced by ovarian stimulated cycle IVF, because it has been believed ovarian stimulated cycle IVF will increase the number of available embryos for transfer. Therefore, it directly increases the chance of pregnancy from the treatment cycle. However, ovarian stimulation is always associated with side effects. The recovery of immature oocytes followed by in-vitro maturation (IVM) and IVF is an attractive alternative to stimulated cycle IVF. IVM treatment provides a successful option to infertile women with polycystic ovaries and polycystic ovary syndrome. It is now possible to combine natural cycle IVF with IVM as an alternative for a selected group of women with various causes of infertility without recourse to ovarian stimulation.     

超排卵周期未成熟卵体外培养的研究

目的:研究来源于超排卵周期中的未成熟卵在拆除卵丘细胞后进行体外成熟培养(IVM)的成熟、受精及胚胎发育能力,探讨IVM技术的临床应用。方法:选取46名体外受精/卵胞浆内单精子显微注射-胚胎移植(IVF/ICSI-ET)患者为研究对象,比较MI和GV期不成熟卵的体外成熟情况,并比较体内成熟卵和体外成熟卵进行ICSI后的正常受精、异常受精、卵裂和优质胚胎形成情况。结果:体外培养中69.8%的MI期卵和77.2%的GV期卵均在24小时内达到成熟,其24小时和48小时的成熟率、总成熟率均无明显差异(P>0.05)。体外成熟卵与体内成熟卵相比较,正常受精率、异常受精率和卵裂率均无明显差异(P>0.05),优质胚胎形成率较低,差异有显著性(P<0.05)。结论:常规超排卵周期中的未成熟卵在拆除卵丘细胞后能够继续体外发育成熟,具有与体内成熟卵相似的ICSI受精、卵裂能力。虽然优质胚胎的形成率低于体内成熟卵,但增加了可移植胚胎和冷冻胚胎数量,提高了助孕成功率。     

How old is too old for in vitro maturation (IVM) treatment?

ObjectiveFertility declines with age. A study of the outcomes of in vitro maturation (IVM) in women of different ages has not been reported to date. The aim of our study was to evaluate the impact of age on treatment response and on pregnancy rates after IVM treatment.Study designWe reviewed the data of all IVM cycles and recorded the total number of oocytes, total metaphase II (MII) oocytes, the number and quality of embryos as well as pregnancy rates. The main outcome measures were number and maturity of retrieved oocytes and pregnancy rates.ResultsCompared to cycles in women ≤25 years, the number of oocytes and total MII oocytes were significantly lower in women over 40 years. The number of good quality embryos in women ≤25 years (3.6 ± 1.7) was higher than in women aged 40 years (0.8 ± 1.2, P < 0.0001). The ongoing pregnancy rates in women aged 20–25 years were 36.8%, 26–35 years were 30.0% and in those 36–39 years were 31.9%. No clinical pregnancy was observed in women over the age of 40.ConclusionWomen over 40 years are poor candidates for IVM treatment.     

Effects of letrozole or tamoxifen coadministered with a standard stimulation protocol on fertility preservation among breast cancer patients

PurposeTo assess the effects of letrozole or tamoxifen coadministration on fertility preservation treatment outcomes.MethodsRetrospective cohort study of 118 breast cancer patients undergoing fertility preservation treatment between 2008 and 2018. Patients who received letrozole (n = 36) or tamoxifen (n = 30) were compared to controls (n = 52) who underwent standard ovarian stimulation protocols. The primary outcome measures included the number of retrieved oocytes, mature oocytes (MII), fertilization, and top-quality embryo rates. The secondary outcome measures included duration of stimulation, gonadotropin dose and peak estradiol level.ResultsThe number of oocytes retrieved, MII oocytes, fertilization rate, duration of stimulation, or gonadotropin dose were similar in the letrozole and tamoxifen groups, compared to controls. Top-quality embryo rate was lower in the tamoxifen group compared to controls (25% vs 39.4%, respectively, P = 0.034). The abnormal fertilization rate was higher in the letrozole group compared to controls (7.8% vs 3.60%, respectively, P = 0.015). A stepwise logistic regression analysis revealed that letrozole and peak estradiol were significantly associated with abnormal fertilization (OR 11.94; 95% CI 2.35–60.4, P = 0.003 for letrozole and OR 1.075; 95% CI 1.024–1.12, P = 0.004 per 100 unit change in estradiol).ConclusionsThere may be a negative effect of letrozole or tamoxifen on fertilization and embryo quality, in fertility preservation cycles. Further studies are needed to confirm these findings.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10815-020-02030-y.     

多囊卵巢综合征妇女体外成熟卵母细胞胚胎慢速冷冻复苏移植结局分析

目的评价未成熟卵母细胞体外成熟（IVM）后形成的卵裂期胚胎经慢速冷冻一解冻后的发育能力。方法将2006年1月至2010年12月北京大学第三医院因多囊卵巢综合征（PCOS）合并不孕症行卵裂期胚胎复苏移植的385例患者分为两组：复苏胚胎来源于体外成熟的卵母细胞组（IVM组,46例）和复苏胚胎来源于常规体内成熟的卵母细胞组（IVF组,339例）。采用慢冻速溶法解冻移植后比较两组患者的临床结局。结果IVM组复苏胚胎243枚,复苏后存活162枚,复苏率为66．67％;IVF组复苏胚胎1605枚,复苏后存活1082枚,复苏率为67．41％,两组比较,差异无统计学意义（P〉0．05）。IVM组患者的临床妊娠率和着床率分别为19．30％（11／57）和10．61％（14／132）,明显低于IvF组临床妊娠率（45．45％,175／385）和着床率（26．14％,240／918;P均〈O．05）。结论体外成熟卵母细胞发育形成的卵裂期胚胎慢速冷冻后临床结局欠佳,可能与冻融前胚胎自身的发育潜力有关。     

Timed IVM followed by ICSI in a patient with immature ovarian oocytes

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF-intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)-ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.     

Pre-implantation developmental potential from in vivo and in vitro matured mouse oocytes: a cytoskeletal perspective on oocyte quality

PurposeIn the present study, fertilization and developmental potential of mouse oocytes matured in different conditions were tested. The efficiency of in vitro fertilization (IVF), pre-implantation development and some important aspects of cytokinesis during early cleavages are discussed.MethodsIn vivo matured (IVO), in vitro matured (IVM) and roscovitine-treated (IVM-Rosco) mouse oocytes were subjected to IVF under identical conditions. Three replicates per group were analyzed. Fertilization was identified by the presence of two pronuclei at 6–8 h post-fertilization. Evaluation of pre-implantation embryonic development was done daily from day 2 to day 5 and embryos were processed for analyses of chromatin, nuclear lamina, microtubules and centrosomal proteins by conventional and confocal fluorescence microscopy.ResultsBoth IVM groups displayed lower fertilization rates when compared to in vivo controls. While IVO-derived embryos exhibit efficient and synchronous progression to the blastocyst stage, both IVM-derived embryos exhibit a delay in embryonic progression, and a lower blastocyst rate. Interestingly, IVM-Rosco M-II oocytes exhibited more blastomere symmetries and higher number of cells at the blastocyst stage than the IVM group with the most notable influence being on the centrosome-microtubule complex of blastomeres.ConclusionOur study strongly indicates that when compared to spontaneously in vitro matured oocytes, treatment with roscovitine may partially enhance developmental competence by maintaining coordination between nuclear and cytoplasmic events. Further evidence is given of cytoskeletal biomarkers that can be identified during in vitro oocyte maturation conditions.     

Laparoscopic excision of ovarian endometrioma does not exert a qualitative effect on ovarian function: insights from in vitro fertilization and single embryo transfer cycles

Purpose

To evaluate whether laparoscopic excision of endometrioma exerts a qualitative effect on ovarian function.

Methods

A retrospective analysis of oocytes retrieved in 25 cycles of 21 patients undergoing IVF treatment with controlled ovarian stimulation. The number of oocytes recovered from ovaries with a history of excision of endometrioma (E-Ov) were compared to those from contra-lateral healthy ovaries (H-Ov) as for the analysis of a quantitative effect of surgery. As for the analysis of a qualitative effect, 55 oocytes from E-Ov were compared to 128 oocytes from H-Ov in terms of normal fertilization rate and the rate of top-quality embryos per normally fertilized eggs. Furthermore, 10 embryos derived from oocytes recovered from E-Ov were compared to 24 embryos derived from oocytes from H-Ov in terms of clinical and on-going pregnancy rates per embryos in 34 single embryo transfer cycles.

Results

Mean number of oocytes recovered from E-Ov was significantly smaller than that from H-Ov (2.2 ± 2.0 vs. 5.1 ± 3.3, P = 0.009). There was no difference between oocytes from E-Ov and H-Ov as for normal fertilization rate (63.6 % vs. 69.5 %, P = 0.43) and the rate of top-quality embryos (40.0 % vs. 49.0 %, P = 0.34). Clinical and on-going pregnancy rates per embryos were also similar in embryos derived from oocytes recovered from E-Ov and H-Ov (40.0 % vs. 25.0 %, P = 0.39 and 20.0 % vs. 20.8 %, P = 0.96).

Conclusions

The quality of oocytes recovered from the ovary with a history of laparoscopic excision of endometrioma is not inferior to the quality of oocytes from contra-lateral healthy ovary.     

Morphokinetics of embryos developed from oocytes matured in vitro

Purpose

In in vitro maturation (IVM) cycles primed with human chorionic gonadotropin (hCG), both immature and mature oocytes are retrieved from antral follicles sized 8–12 mm. Using time-lapse microscopy, we compared the morphokinetic behavior of embryos developed from oocytes matured in vivo and in vitro, testing the hypothesis that IVM affects preimplantation development. Furthermore, we extended the morphokinetic analysis of these embryos by a comparison with embryos obtained in stimulated assisted reproduction technology (ART) cycles.

Methods

In IVM cycles primed with follicle-stimulating hormone (FSH)/hCG, prior to sperm microinjection, oocytes surrounded by an expanded cumulus at retrieval and presumably mature (EC-MII) were incubated for 6 h, while immature oocytes enclosed in a compact cumulus (CC) were matured in vitro for 30 h. The morphokinetics of embryos selected for transfer or cryopreservation, derived from EC-MII and CC oocytes, were comparatively and retrospectively analyzed in terms of cleavage times (t2, t3, t4, t5, and t8) and intervals (cc2, cc3, s2, s3). For further comparison, the morphokinetics of embryos selected for transfer or cryopreservation (ICSI) or giving rise to ongoing pregnancies (model) in stimulated ART cycles was also assessed.

Results

The morphokinetic behavior of EC-MII and CC embryos was entirely comparable, as suggested by the absence of statistical differences in the averages of all cleavage times and intervals. Almost all cleavage and interval times were also similar between EC-MII, CC, ICSI, and model groups, with the exception of t4 and s2, which were delayed and longer, respectively, in embryos generated in IVM cycles (EC-MII and CC).

Conclusions

These findings do not support the hypothesis that maturation in vitro affects embryo morphokinetics, while they suggest only marginal differences in the morphokinetics of embryos developed from oocytes matured in vivo and in vitro in IVM cycles and embryos developed from mature oocytes recovered in stimulated cycles.