DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. The causes of this DNA damage are still uncertain but the major candidates are oxidative stress and aberrant apoptosis. The weight of evidence currently favours the former and, in keeping with this conclusion, positive results have been reported for antioxidant therapy both in vivo and in vitro. Resolving the causes of DNA damage in the male germ line will be essential if we are to prevent the generation of genetically damaged human embryos, particularly in the context of assisted conception therapy.

DNA damage to spermatozoa has impacts on fertilization and pregnancy

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.

/pdf/sperm-preparation-for-art-22vrp3ut6i.pdf

Sperm preparation for ART.

Background
Progesterone prepares the endometrium for pregnancy by stimulating proliferation in response to human chorionic gonadotropin (hCG) produced by the corpus luteum in the luteal phase of the menstrual cycle. In assisted reproduction techniques (ART), progesterone and/or hCG levels are low, so the luteal phase is supported with progesterone, hCG or gonadotropin-releasing hormone (GnRH) agonists to improve implantation and pregnancy rates.

Objectives
To determine the relative effectiveness and safety of methods of luteal phase support provided to subfertile women undergoing assisted reproduction.

Search methods
We searched databases including the Cochrane Menstrual Disorders and Subfertility Group (MDSG) Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, PsycINFO and trial registers up to November 2014. Further searches were undertaken in August 2015.

Selection criteria
Randomised controlled trials (RCTs) of luteal phase support using progesterone, hCG or GnRH agonist supplementation in ART cycles.

Data collection and analysis
We used standard methodological procedures expected by Cochrane. Our primary outcome was live birth or ongoing pregnancy. The overall quality of the evidence was assessed using GRADE methods.

Main results
Ninety-four RCTs (26,198 women) were included. Most studies had unclear or high risk of bias in most domains. The main limitations in the evidence were poor reporting of study methods and imprecision due to small sample sizes.

1. hCG vs placebo/no treatment (five RCTs, 746 women)

Findings suggested benefit for the hCG group in live birth or ongoing pregnancy rates when data were analysed with a fixed-effect model (OR 1.76, 95% CI 1.08 to 2.86, three RCTs, 527 women, I2 = 24%, very low-quality evidence) but there was no clear evidence of a difference using a random-effects model (OR 1.67, 95% CI 0.90 to 3.12). hCG may increase ovarian hyperstimulation syndrome (OHSS) rates (OR 4.28, 95% CI 1.91 to 9.6, one RCT, 387 women, low-quality evidence).

2. Progesterone vs placebo/no treatment (eight RCTs, 875 women)

Findings suggested benefit for the progesterone group in live birth or ongoing pregnancy rates when data were analysed with a fixed-effect model (OR 1.77, 95% CI 1.09 to 2.86, five RCTs, 642 women, I2 = 35%, very low-quality evidence) but there was no clear evidence of a difference using a random-effects model (OR 1.77, 95% CI 0.96 to 3.26). OHSS was not reported.

3. Progesterone vs hCG regimens (16 RCTs, 2162 women)

hCG regimens included hCG alone and hCG with progesterone. There was no evidence of a difference between progesterone and hCG regimens in live birth or ongoing pregnancy rates (OR 0.95, 95% CI 0.65 to 1.38, five RCTs, 833 women, I2 = 0%, low-quality evidence). Progesterone was associated with lower OHSS rates than hCG regimens (OR 0.46, 95% CI 0.30 to 0.71, 5 RCTs, 1293 women , I2=48%).

4. Progesterone vs progesterone with oestrogen (16 RCTs, 2577 women)

There was no evidence of a difference between the groups in rates of live birth or ongoing pregnancy (OR 1.12, 95% CI 0.91 to 1.38, nine RCTs, 1651 women, I2 = 0%, low-quality evidence) or OHSS (OR 0.56, 95% CI 0.2 to 1.63, two RCTs, 461 women, I2 = 0%, low-quality evidence).

5. Progesterone vs progesterone + GnRH agonist (seven RCTs, 1708 women)

Live birth or ongoing pregnancy rates were lower in the progesterone-only group than the progesterone plus GnRH agonist group (OR 0.62, 95% CI 0.48 to 0.81, nine RCTs, 2861 women, I2 = 55%, random effects, low-quality evidence). Statistical heterogeneity was high but the direction of effect was consistent across studies. OHSS was reported in one study only; there was no evidence of a difference between the groups (OR 1.00, 95% CI 0.33 to 3.01, one RCT, 300 women, very low quality evidence).

6. Progesterone regimens (45 RCTs, 13,814 women)

There were nine different comparisons between progesterone regimens. Findings for live birth or ongoing pregnancy were as follows: intramuscular (IM) versus oral: OR 0.71, 95% CI 0.14 to 3.66 (one RCT, 40 women, very low-quality evidence); IM versus vaginal/rectal: OR 1.37, 95% CI 0.94 to 1.99 (seven RCTs, 2309 women, I2 = 71%, random effects, very low-quality evidence); vaginal/rectal versus oral: OR 1.19, 95% CI 0.83 to 1.69 (four RCTs, 857 women, I2 = 32%, low-quality evidence); low-dose versus high-dose vaginal: OR 0.97, 95% CI 0.84 to 1.11 (five RCTs, 3720 women, I2 = 0%, moderate-quality evidence); short versus long protocol: OR 1.04, 95% CI 0.79 to 1.36 (five RCTs, 1205 women, I2 = 0%, low-quality evidence); micronised versus synthetic: OR 0.9, 95% CI 0.53 to 1.55 (two RCTs, 470 women, I2 = 0%, low-quality evidence); vaginal ring versus gel: OR 1.09, 95% CI 0.88 to 1.36 (one RCT, 1271 women, low-quality evidence); subcutaneous versus vaginal gel: OR 0.92, 95% CI 0.74 to 1.14 (two RCTs, 1465 women, I2 = 0%, low-quality evidence); vaginal versus rectal: OR 1.28, 95% CI 0.64 to 2.54 (one RCT, 147 women, very low-quality evidence). OHSS rates were reported for only two comparisons: IM versus oral, and low versus high-dose vaginal; there was no evidence of a difference between the groups.

7. Progesterone and oestrogen regimens (two RCTs, 1195 women)

The included studies compared two different oestrogen protocols. There was no evidence of a difference in live birth or ongoing pregnancy rates between a short or long protocol (OR 1.08, 95% CI 0.81 to 1.43, one RCT, 910 women, low-quality evidence) or between a low or high dose of oestrogen (OR 0.65, 95% CI 0.37 to 1.13, one RCT, 285 women, very low-quality evidence). Neither study reported OHSS.

Authors' conclusions
hCG or progesterone given during the luteal phase may be associated with higher rates of live birth or ongoing pregnancy than placebo or no treatment, but the evidence is not conclusive. The addition of GnRHa to progesterone appears to improve outcomes. hCG may increase the risk of OHSS compared to placebo. Moreover hCG, with or without progesterone, is associated with higher rates of OHSS than progesterone alone. Neither the addition of oestrogen nor the route of progesterone administration appears to be associated with an improvement in outcomes.

/pdf/luteal-phase-support-for-assisted-reproduction-cycles-4s2nd8fmnb.pdf

Luteal phase support for assisted reproduction cycles

Background The aspiration of the granulosa cells that surround the oocyte and the use of gonadotropin releasing hormone agonists (GnRHa) during assisted reproduction technology (ART) treatment can interfere with the production, during the luteal phase, of progesterone, which is necessary for successful implantation of the embryo. Providing hormonal supplementation during the luteal phase with either progesterone itself, or human chorionic gonadotropin (hCG), which stimulates progesterone production, may improve implantation and, thus, pregnancy rates. Objectives To determine (1) if luteal phase support after assisted reproduction increases the pregnancy rate, (2) the optimal hormone for luteal phase support, i.e. hCG, progesterone, or a combination of both, and (3) the optimal route of progesterone administration. Search strategy We searched the Cochrane Menstrual Disorders & Subfertility Group trials register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (1971 to Dec 2003), EMBASE (1985 to Dec 2003). We handsearched reference lists of relevant articles were scanned, and abstract books from scientific meetings up to December 2003. Selection criteria Randomized controlled trials of luteal phase support after ART treatment, comparing hCG or progesterone with placebo or no treatment, comparing progesterone with hCG, progesterone plus hCG, or progesterone plus estrogen, or comparing different routes of progesterone administration. Quasi-randomized trials were excluded from the main analyses, but included in a secondary analysis for each comparison. Data collection and analysis For each comparison, data on live birth, ongoing and clinical pregnancy per embryo or gamete transfer procedure, miscarriage per clinical pregnancy, ovarian hyperstimulation syndrome (OHSS) per transfer, and multiple pregnancy per clinical pregnancy were extracted into 2 x 2 tables and subgrouped by use of GnRHa in the ovarian stimulation regimen. The odds ratio (OR) and risk difference (RD) were calculated. Main results Fifty-nine studies were included in the review. Luteal phase support with hCG provided significant benefit, compared to placebo or no treatment, in terms of increased ongoing pregnancy rates (odds ratio (OR) 2.38, 95% confidence interval (CI) 1.32 to 4.29) and decreased miscarriage rates (OR 0.12, 95% CI 0.03 to 0.50), but only when GnRHa was used. The odds of OHSS increased 20-fold when hCG was used in cycles with GnRHa. Progesterone use resulted in a small but significant increase in pregnancy rates (OR 1.34, 95% CI 1.01 to 1.79) when trials with and without GnRHa were grouped together, but no effect on the miscarriage rate was observed. No significant difference was found between progesterone and hCG or between progesterone and progesterone plus hCG or estrogen in terms of pregnancy or miscarriage rates, but the odds of OHSS were more than 2-fold higher with treatments involving hCG than with progesterone alone(OR 3.06, 95% CI 1.59 to 5.86). Comparing routes of progesterone administration, reductions in clinical pregnancy rate with the oral route, compared to the intramuscular or vaginal routes, did not reach statistical significance, but there was evidence of benefit of the intramuscular over the vaginal route for the outcomes of ongoing pregnancy and live birth. No significant difference in pregnancy rate was observed between vaginal progesterone gel and other types of vaginal progesterone. Reviewers' conclusions Luteal phase support with hCG or progesterone after assisted reproduction results in an increased pregnancy rate. hCG does not provide better results than progesterone, and is associated with a greater risk of OHSS when used with GnRHa. The optimal route of progesterone administration has not yet been established.

Luteal phase support in assisted reproduction cycles

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mrd.1080370314

Molecular biology of human male infertility: Links with aging, mitochondrial genetics, and oxidative stress?

Influence of pentoxifylline in severe male factor infertility.

The relative chance of pregnancy following tubal or uterine transfer procedures.

Preliminary results using pentoxifylline in a pronuclear stage tubal transfer (PROST) program for severe male factor infertility.

Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study

The present report describes the motility changes in vitro (percentage motile and progressively motile) of freshly collected testicular and epididymal spermatozoa and following freeze/thaw of the same spermatozoa from a man with obstructive azoospermia. Washed spermatozoa were cultured in micro droplets under paraffin oil or in test tubes using HEPES-buffered or bicarbonate-buffered medium containing 10% human serum. In fresh testicular sperm cultures 60-65% of the sperm cells became motile within 2 days of culture ; the motility was maintained for a further 4-5 days before a decline was observed. The progressive motility improved markedly on the third day of culture and it peaked around day 5. Only a small number of frozen/thawed testicular spermatozoa became motile during in-vitro culture (15-20%) and the motility was maintained for only 2-3 days before it declined. Furthermore, only 10-12% of the spermatozoa showed progressive motility. Spermatozoa recovered from micro-epididymal sperm aspiration (MESA) showed a gradual decrease in progressive motility and in 5 days all sperm cells were found to be immotile in both freshly collected and frozen/thawed spermatozoa. All culture systems supported sperm motility. It is clear that testicular spermatozoa, particularly from men with obstructive azoospermia, can be collected and maintained in vitro for up to 1 week before the oocyte retrieval but when frozen testicular or epididymal spermatozoa are used it is more reliable to thaw these spermatozoa on the day of intracytoplasmic sperm injection.

Case report: Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection

W. Rohini Edirisinghe

Papers

Influence of pentoxifylline in severe male factor infertility.

The relative chance of pregnancy following tubal or uterine transfer procedures.

Preliminary results using pentoxifylline in a pronuclear stage tubal transfer (PROST) program for severe male factor infertility.

Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study

Case report: Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection