Background
Advances in cell culture media have led to a shift in in vitro fertilisation (IVF) practice from cleavage stage embryo transfer to blastocyst stage transfer. The rationale for blastocyst transfer is to improve both uterine and embryonic synchronicity and enable self selection of viable embryos, thus resulting in better live birth rates.

Objectives
To determine whether blastocyst stage (day 5 to 6) embryo transfers improve the live birth rate, and other associated outcomes, compared with cleavage stage (day 2 to 3) embryo transfers.

Search methods
We searched the Cochrane Gynaecology and Fertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL; the Cochrane Library; 2016, Issue 4), MEDLINE, EMBASE, PsycINFO, CINAHL, and Bio extracts from inception to 4th April 2016. We also searched registers of ongoing trials and the reference lists of studies retrieved.

Selection criteria
We included randomised controlled trials (RCTs) which compared the effectiveness of blastocyst versus cleavage stage transfers.

Data collection and analysis
We used standard methodological procedures recommended by Cochrane. Our primary outcomes were live birth and cumulative clinical pregnancy rates. Secondary outcomes were clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos, and embryo freezing. We assessed the overall quality of the evidence for the main comparisons using GRADE methods.

Main results
We included 27 RCTs (4031 couples or women).

The live birth rate following fresh transfer was higher in the blastocyst transfer group (odds ratio (OR) 1.48, 95% confidence interval (CI) 1.20 to 1.82; 13 RCTs, 1630 women, I2 = 45%, low quality evidence) following fresh transfer. This suggests that if 29% of women achieve live birth after fresh cleavage stage transfer, between 32% and 42% would do so after fresh blastocyst stage transfer.

There was no evidence of a difference between the groups in rates per couple of cumulative pregnancy following fresh and frozen-thawed transfer after one oocyte retrieval (OR 0.89, 95% CI 0.64 to 1.22; 5 RCTs, 632 women, I2 = 71%, very low quality evidence).

The clinical pregnancy rate was also higher in the blastocyst transfer group, following fresh transfer (OR 1.30, 95% CI 1.14 to 1.47; 27 RCTs, 4031 women, I2 = 56%, moderate quality evidence). This suggests that if 36% of women achieve clinical pregnancy after fresh cleavage stage transfer, between 39% and 46% would do so after fresh blastocyst stage transfer.

There was no evidence of a difference between the groups in rates of multiple pregnancy (OR 1.05, 95% CI 0.83 to 1.33; 19 RCTs, 3019 women, I2 = 30%, low quality evidence), or miscarriage (OR 1.15, 95% CI 0.88 to 1.50; 18 RCTs, 2917 women, I2 = 0%, low quality evidence). These data are incomplete as under 70% of studies reported these outcomes.

Embryo freezing rates were lower in the blastocyst transfer group (OR 0.48, 95% CI 0.40 to 0.57; 14 RCTs, 2292 women, I2 = 84%, low quality evidence). This suggests that if 60% of women have embryos frozen after cleavage stage transfer, between 37% and 46% would do so after blastocyst stage transfer. Failure to transfer any embryos was higher in the blastocyst transfer group (OR 2.50, 95% CI 1.76 to 3.55; 17 RCTs, 2577 women, I2 = 36%, moderate quality evidence). This suggests that if 1% of women have no embryos transferred in (planned) fresh cleavage stage transfer, between 2% and 4% will have no embryos transferred in (planned) fresh blastocyst stage transfer.

The evidence was of low quality for most outcomes. The main limitation was serious risk of bias, associated with failure to describe acceptable methods of randomisation, and unclear or high risk of attrition bias.

Authors' conclusions
There is low quality evidence for live birth and moderate quality evidence for clinical pregnancy that fresh blastocyst stage transfer is associated with higher rates than fresh cleavage stage transfer. There was no evidence of a difference between the groups in cumulative pregnancy rates derived from fresh and frozen-thawed cycles following a single oocyte retrieval, but the evidence for this outcome was very low quality. Thus, although there is a benefit favouring blastocyst transfer in fresh cycles, it remains unclear whether the day of transfer impacts on cumulative live birth and pregnancy rates. Future RCTs should report rates of live birth, cumulative live birth, and miscarriage to enable couples or women undergoing assisted reproductive technology (ART) and service providers to make well informed decisions on the best treatment option available.

/pdf/cleavage-stage-versus-blastocyst-stage-embryo-transfer-in-4xyk4mlvlb.pdf

Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology

BACKGROUND: Reactive oxygen species (ROS)-mediated damage to human spermatozoa has been implicated in the pathogenesis of male infertility. Although ROS production by human spermatozoa has been extensively studied, the cell-to-cell variation in ROS production by spermatozoa at different stages of maturation has never been investigated. METHODS: In this study, we determined ROS production by subsets of human spermatozoa at different stages of maturation isolated by density gradient centrifugation of ejaculated spermatozoa obtained from healthy donors and from patients attending a clinic for infertility screening. RESULTS: Four different fractions were obtained. ROS production was highest in immature spermatozoa with abnormal head morphology and cytoplasmic retention and lowest in mature spermatozoa and immature germ cells (P < 0.01). ROS production was highest in immature spermatozoa from males with abnormal semen parameters compared with donors (P < 0.0001) or patients with normal semen parameters (P 0.015). ROS production by immature spermatozoa was inversely correlated with the recovery of motile, mature spermatozoa in the high density fraction 4 (P 0.01). CONCLUSIONS: The results of this study indicate that there is significant cell-to-cell variation in ROS production in subsets of spermatozoa at different stages of maturation and that oxidative damage of mature spermatozoa by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis may be an important cause of male infertility.

/pdf/differential-production-of-reactive-oxygen-species-by-52a2uw8gce.pdf

Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation

Background Despite numerous advances in the field of in vitro fertilisation (IVF), many of the widely applied embryo culture techniques and resulting implantation rates have remained relatively unchanged since the first treatment was performed in the mid 1970's. Recent advances in the understanding of nutrient requirements of embryos, have led to a renaissance of extending their culture from the standard procedure of 2-3 days (early cleavage embryo transfer) to 5-6 days (blastocyst culture). The rationale for blastocyst culture is to improve the synchronicity of uterine and embryonic development and provide a mechanism for self-selection of viable embryos. Numerous reports on the clinical benefits of blastocyst culture have led to the worldwide introduction of this technique, despite a deficiency of conclusive evidence to do so. Objectives Primary: To determine if blastocyst stage embryo transfers (ET's) result in higher success rates, than cleavage stage embryo transfers. Secondary: To assess the overall embryo utilisation rate of both techniques. Search strategy Electronic searches of the Cochrane Menstrual Disorders and Subfertility Group specialised register of controlled trials, CCTR, MEDLINE, EMBASE, and Bio extracts were performed to identify relevant randomised controlled trials (RCTs). Attempts were also made to identify trials from the National Research Register, the Clinical Trial Register and the citation lists of review articles and included trials. The first or corresponding author of each included trial was also contacted for additional information. Selection criteria Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers. Data collection and analysis Of the 29 trials that were identified, ten trials met the inclusion criteria and were reviewed. Primary outcomes were rates of; live birth, clinical pregnancy and implantation per woman. Secondary outcomes were rates of; miscarriage, monozygotic twinning, embryo freezing, embryo utilisation, cancellation, multiple pregnancy and high order pregnancy and per cycle data. Quality assessment and data extraction were performed independently by two reviewers. Meta analysis was performed using odds ratios for dichotomous outcomes and weighted mean differences for continuous outcomes. Main results There was no significant difference between the two treatment groups in live birth rate, although this was reported by only one quasi-random trial (Peto OR 1.59, 95% CI 0.80, 3.15). There was also no evidence of a difference in pregnancy rate (both overall and subgroups) between the two groups for pregnancy rate per couple randomised (4 RCTs: Peto OR 0.86, 95% CI 0.57, 1.29). There was also no suggestion of an overall difference in implantation rates per embryo's transferred although it was impossible to calculate valid confidence intervals from published data (Day 2/3 17.1% vs Day 5/6 18.9%). The subgroup of sequential media trials suggested higher implantation rate for blastocyst transfer (Day 2/3 22.6% vs Day 5/6 32%). The miscarriage rate was no different between the two groups (1 RCT, Peto OR 1.66, 95% CI 0.41, 6.81). The RCTs reporting embryo freezing showed no difference (Peto OR 1.71, 95% CI 1.00, 2.94), however the two quasi-random trials showed a significant difference in favour of the Day 2/3 group (Peto OR 2.99, 95% CI 1.88, 4.75). Embryo transfer cancellation rates were significantly higher in the Day 5/6 group (5 RCTs: Peto OR 0.57, 95% CI 0.40, 0.83). There was no significant difference in the rate of multiple pregnancies or the rate of high order pregnancies (3 RCTs, Peto OR 0.58, 95% CI 0.30, 1.12)(2 RCTs, Peto OR 7.88, 95% CI 0.49, 126.30 respectively). Reviewer's conclusions Overall this review of the best available evidence based on data from randomised controlled trials, suggests that to date little difference in the major outcome parameters has been demonstrated between early embryo transfer and blastocyst culture. Collectively, the increase in cancellation and the possible decrease in cryopreservation rates suggest that the routine practice of blastocyst culture should be offered to patients with caution. The subgroup of trials employing sequential media, did however demonstrate a substantial improvement in implantation rates and similar pregnancy rates, despite the transfer of less embryos. Whether this trend will culminate in convincing higher live birth rates per woman, has yet to be validated.

Cleavage stage versus blastocyst stage embryo transfer in assisted conception.

Background
Among subfertile couples undergoing assisted reproductive technology (ART), pregnancy rates following frozen-thawed embryo transfer (FET) treatment cycles have historically been found to be lower than following embryo transfer undertaken two to five days following oocyte retrieval. Nevertheless, FET increases the cumulative pregnancy rate, reduces cost, is relatively simple to undertake and can be accomplished in a shorter time period than repeated in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) cycles with fresh embryo transfer. FET is performed using different cycle regimens: spontaneous ovulatory (natural) cycles; cycles in which the endometrium is artificially prepared by oestrogen and progesterone hormones, commonly known as hormone therapy (HT) FET cycles; and cycles in which ovulation is induced by drugs (ovulation induction FET cycles). HT can be used with or without a gonadotrophin releasing hormone agonist (GnRHa). This is an update of a Cochrane review; the first version was published in 2008.

Objectives
To compare the effectiveness and safety of natural cycle FET, HT cycle FET and ovulation induction cycle FET, and compare subtypes of these regimens.

Search methods
On 13 December 2016 we searched databases including Cochrane Gynaecology and Fertility's Specialised Register, CENTRAL, MEDLINE, Embase, PsycINFO and CINAHL. Other search sources were trials registers and reference lists of included studies.

Selection criteria
We included randomized controlled trials (RCTs) comparing the various cycle regimens and different methods used to prepare the endometrium during FET.

Data collection and analysis
We used standard methodological procedures recommended by Cochrane. Our primary outcomes were live birth rates and miscarriage.

Main results
We included 18 RCTs comparing different cycle regimens for FET in 3815 women. The quality of the evidence was low or very low. The main limitations were failure to report important clinical outcomes, poor reporting of study methods and imprecision due to low event rates. We found no data specific to non-ovulatory women.

1. Natural cycle FET comparisons

Natural cycle FET versus HT FET

No study reported live birth rates, miscarriage or ongoing pregnancy.

There was no evidence of a difference in multiple pregnancy rates between women in natural cycles and those in HT FET cycle (odds ratio (OR) 2.48, 95% confidence interval (CI) 0.09 to 68.14, 1 RCT, n = 21, very low-quality evidence).

Natural cycle FET versus HT plus GnRHa suppression

There was no evidence of a difference in rates of live birth (OR 0.77, 95% CI 0.39 to 1.53, 1 RCT, n = 159, low-quality evidence) or multiple pregnancy (OR 0.58, 95% CI 0.13 to 2.50, 1 RCT, n = 159, low-quality evidence) between women who had natural cycle FET and those who had HT FET cycles with GnRHa suppression. No study reported miscarriage or ongoing pregnancy.

Natural cycle FET versus modified natural cycle FET (human chorionic gonadotrophin (HCG) trigger)

There was no evidence of a difference in rates of live birth (OR 0.55, 95% CI 0.16 to 1.93, 1 RCT, n = 60, very low-quality evidence) or miscarriage (OR 0.20, 95% CI 0.01 to 4.13, 1 RCT, n = 168, very low-quality evidence) between women in natural cycles and women in natural cycles with HCG trigger. However, very low-quality evidence suggested that women in natural cycles (without HCG trigger) may have higher ongoing pregnancy rates (OR 2.44, 95% CI 1.03 to 5.76, 1 RCT, n = 168). There were no data on multiple pregnancy.

2. Modified natural cycle FET comparisons

Modified natural cycle FET (HCG trigger) versus HT FET

There was no evidence of a difference in rates of live birth (OR 1.34, 95% CI 0.88 to 2.05, 1 RCT, n = 959, low-quality evidence) or ongoing pregnancy (OR 1.21, 95% CI 0.80 to 1.83, 1 RCT, n = 959, low-quality evidence) between women in modified natural cycles and those who received HT. There were no data on miscarriage or multiple pregnancy.

Modified natural cycle FET (HCG trigger) versus HT plus GnRHa suppression

There was no evidence of a difference between the two groups in rates of live birth (OR 1.11, 95% CI 0.66 to 1.87, 1 RCT, n = 236, low-quality evidence) or miscarriage (OR 0.74, 95% CI 0.25 to 2.19, 1 RCT, n = 236, low-quality evidence) rates. There were no data on ongoing pregnancy or multiple pregnancy.

3. HT FET comparisons

HT FET versus HT plus GnRHa suppression

HT alone was associated with a lower live birth rate than HT with GnRHa suppression (OR 0.10, 95% CI 0.04 to 0.30, 1 RCT, n = 75, low-quality evidence). There was no evidence of a difference between the groups in either miscarriage (OR 0.64, 95% CI 0.37 to 1.12, 6 RCTs, n = 991, I2 = 0%, low-quality evidence) or ongoing pregnancy (OR 1.72, 95% CI 0.61 to 4.85, 1 RCT, n = 106, very low-quality evidence).

There were no data on multiple pregnancy.

4. Comparison of subtypes of ovulation induction FET

Human menopausal gonadotrophin(HMG) versus clomiphene plus HMG

HMG alone was associated with a higher live birth rate than clomiphene combined with HMG (OR 2.49, 95% CI 1.07 to 5.80, 1 RCT, n = 209, very low-quality evidence). There was no evidence of a difference between the groups in either miscarriage (OR 1.33, 95% CI 0.35 to 5.09,1 RCT, n = 209, very low-quality evidence) or multiple pregnancy (OR 1.41, 95% CI 0.31 to 6.48, 1 RCT, n = 209, very low-quality evidence).

There were no data on ongoing pregnancy.

Authors' conclusions
This review did not find sufficient evidence to support the use of one cycle regimen in preference to another in preparation for FET in subfertile women with regular ovulatory cycles. The most common modalities for FET are natural cycle with or without HCG trigger or endometrial preparation with HT, with or without GnRHa suppression. We identified only four direct comparisons of these two modalities and there was insufficient evidence to support the use of either one in preference to the other.

Cycle regimens for frozen-thawed embryo transfer

https://www.clevelandclinic.org/reproductiveresearchcenter/docs/agradoc134.pdf

Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index

Two techniques for the separation of spermatozoa were compared: swim-up migration (SUM) and centrifugation on a discontinuous Percoll gradient (CPG). Their respective effects on sperm motility were analysed by computer-assisted videomicrography in either normal or asthenozoospermic groups. In both groups, there was no difference in any of the motion parameters between the two treatments after 1-h incubation. However, a clear difference was observed after 24 h when excellent motility was retained only in the CPG-treated group. A total of 350 ejaculates were produced by the husbands of women undergoing oocyte retrieval in an IVF programme. Spermatozoa were treated by CPG when the infertility was due to poor quality spermatozoa (n = 91), when there was a known previous history of semen infection (n = 73) or when frozen semen, originating from a donor, was used (n = 36). In all other cases (n = 150), spermatozoa were treated by SUM. The cleavage rates obtained were 32.2, 70.1, 60.9 and 68.6% respectively in the four categories. The clinical pregnancy rates per oocyte retrieval were 19.8, 31.5, 22.2 and 18.0% respectively. Forty-eight births occurred in the CPG group: 28 boys and 20 girls, all normal. We conclude that CPG is useful, both in cases of poor semen quality and in tubal infertility, in which the clinical pregnancy rate increased significantly from 18.0 to 31.5%.

Improvement of survival and fertilizing capacity of human spermatozoa in an IVF programme by selection on discontinuous Percoll gradients

A total of 130 transfers of frozen-thawed (F-T) human embryos was carried out after moderate ovarian stimulation with human menopausal gonadotrophin (HMG). Embryos were replaced 3 days after the spontaneous luteinizing hormone (LH) surge or 4 days if ovulation was induced by human chorionic gonadotrophin (HCG). Embryos were thawed a few hours prior to transfer. One-hundred-and-twenty-three transfers were effective and 23 pregnancies were achieved. The rate of ongoing pregnancies per transfer was 17.9% (22/123). The survival rate of embryos originating from cycles stimulated by a combination of an LHRH analogue and HMG in a long protocol (LA-HMG protocol) was significantly lower when compared with the rate of embryos retrieved from clomiphene citrate-HMG (CC-HMG protocol) stimulated cycles (52 versus 67%, P less than 0.05). When fresh embryos originated from cycles stimulated with an LHRH analogue and HMG in a short protocol (SA-HMG protocol), the survival rate was not affected (59 versus 67%, NS). Although the difference was not significant, the ongoing pregnancy rate per transfer according to the three protocols from which the embryos originated seemed to be better with the SA-HMG protocol: 16% with the CC-HMG protocol, 14.5% with the LA-HMG protocol versus 27.6% with the SA-HMG protocol. The success rate was independent of the number of F-T transferred embryos if at least one embryo with 100% intact blastomeres was replaced.

Transfers of frozen—thawed human embryos in cycles stimulated by HMG

Male infertility can be successfully treated by In Vitro Fertilization (IVF) and Embryo Transfer. However in case of oligo asthenospermia it is often difficult to select enough spermatozoa with good motility for the oocytes insemination. To cope with this problem, in 27 IVF cycles, sperm selection was performed by centrifugation on discontinuous Percoll density gradient. This method was demonstrated to be more efficient than swim-up migration to select motile sperm. The cleavage rate (63%) and the pregnancy rate per cycle (22%) were both satisfying. Centrifugation on discontinuous Percoll gradient was also found to be highly efficient for the removal of bacterial contaminants from semen (20 IVF cycles); the cleavage rate (66%) and the pregnancy rate per cycle (35%) were also quite satisfying. It can be concluded that centrifugation on discontinuous Percoll gradient is a useful method for sperm preparation in IVF in case of oligo-asthenospermia and in case of bacterial contamination in semen.

[Separation of spermatozoa using Percoll gradients: value for in vitro fertilization].

High-ionic-strength media are known to favor rapid capacitation and acrosome reaction of spermatozoa in vitro. The influence of isoosmolar (N-BWW) and hyperosmolar (H-BWW) media on the percent motility, forward progression, and zona-free hamster egg penetration has been investigated for 148 semen from patients consulting for male or idiopathic sterility. After 15 h of incubation, the hyperosmotic medium had no significant detrimental effect on the percent motility of spermatozoa, although the forward progression was better maintained in the isoosmolar medium. In general egg penetration was increased when spermatozoa were treated with the hypertonic medium, and in particular 5 patients whose semen showed no penetration when treated with N-BWW scored < 12% when treated with H-BWW. For the 23 subjects who had an IVF at the same period, a good agreement with zona-free egg penetration was found in 70% of the cases.

https://www.tandfonline.com/doi/pdf/10.3109/01485018708988483

In vitro behavior of human nonfertile semen in isoosmotic and hyperosmotic media.

Development of hyperactivated motility is considered as necessary for penetration through zona pellucida. In a study concerning 114 IVF attempts, we could not establish a significant correlation between fertilization rate and hyperactivation rate (HA). On the contrary, most fertilization failures (16/21) we found for HA values < 10%. 10 of such couples could benefit of ICSI for the next attempt: fertilization rates were strictly similar to the general rate observed for ICSI in our group (48%).

M. C. Pinatel

Papers

Improvement of survival and fertilizing capacity of human spermatozoa in an IVF programme by selection on discontinuous Percoll gradients

Transfers of frozen—thawed human embryos in cycles stimulated by HMG

[Separation of spermatozoa using Percoll gradients: value for in vitro fertilization].

In vitro behavior of human nonfertile semen in isoosmotic and hyperosmotic media.

[In vitro fertilization failures related to a sperm hyperactivation defect: indication for microinjection?].