STUDY QUESTION

Can selection of spermatozoa by density gradient centrifugation prior to cryopreservation and/or hypotaurine supplementation improve the post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia?

SUMMARY ANSWER

Sperm selection by density gradient centrifugation before freezing and supplementation of the media by hypotaurine is beneficial for the cryopreservation of semen samples of patients with oligoasthenoteratozoospermia.

WHAT IS KNOWN ALREADY

Sperm from men with oligoasthenoteratozoospermia are more susceptible than normal to cryoinjury. Density gradient centrifugation before sperm freezing may allow the selection of a subpopulation of spermatozoa more resistant to cryopreservation. Hypotaurine is an antioxidant with a protective effect on sperm functions.

STUDY DESIGN, SIZE, DURATION

The experiment was carried out according to a factorial design involving two binary factors resulting in four treatment combinations which were randomly allocated in oligoasthenoteratozoospermia sperm samples from 64 patients recruited between January 2009 and June 2010.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Semen was provided by 64 men undergoing evaluation for infertility at the Centre for Reproductive Medicine of the University Hospital in Clermont-Ferrand, France, between January 2009 and June 2010. Four treatment combinations were tested: sperm freezing before selection without (F-S/H–; n = 16) and with hypotaurine supplementation (F-S/H+; n = 16); sperm selection before freezing without (S-F/H–; n = 16) and with hypotaurine supplementation (S-F/H+; n = 16). Measurements of sperm recovery rates and markers of apoptosis (externalization of phosphatidylserine (PS), mitochondrial membrane potential and DNA fragmentation) were compared in recovered spermatozoa after each procedure.

MAIN RESULTS AND THE ROLE OF CHANCE

Higher recovery rates of progressive and total motile spermatozoa were observed when sperm selection was performed before freezing (P < 0.05). The protective effect of hypotaurine was only observed on the percentage of live spermatozoa with PS externalization among total live spermatozoa (AN+ PI–/((AN+ PI–) + (AN– PI–)) when the sperm selection by density gradient centrifugation was performed before freezing (S-F/H+ versus S-F/H–: 6.8 ± 1.09 versus 11.8 ± 2.03%, P = 0.04). The percentage of mitochondrial membrane potential (DiOC₆(3) ^high) spermatozoa was higher (P = 0.001) when sperm selection was done before freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 58.1 ± 3.50 versus 46.7 ± 5.48%) or without (S-F/H– versus F-S/H–: 57.0 ± 5.18 versus 35.4 ± 4.99%) hypotaurine supplementation. The percentages of TUNEL+ spermatozoa were significantly lower (P = 0.001) when sperm selection was done before sperm freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 38.6 ± 9.59 versus 55.7 ± 5.88%) or without hypotaurine supplementation (S-F/H– versus F-S/H–: 37.2 ± 7.91 versus 71.0 ± 5.66%).

LIMITATIONS, REASONS FOR CAUTION

The ICSI outcomes were not assessed and the fertility of the spermatozoa remains unknown.

WIDER IMPLICATIONS OF THE FINDINGS

Sperm selection by density gradient centrifugation before freezing and hypotaurine supplementation could improve the cryopreservation of sperm from oligoasthenoteratozoospermic men and make a larger number of functional spermatozoa available for ICSI.

STUDY FUNDING/COMPETING INTERETS(S)

This work was supported by a hospital grant (Projet Hospitalier Recherche Clinique, CHU Clermont Ferrand, France). None of the authors has any conflict of interest to declare.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/8/2045?rss=1

Recommendation and review posted by G. Smith

STUDY QUESTION

What are the levels of anandamide (N-arachidonoylethanolamide, AEA) in human seminal plasma and how are these related to abnormal spermatozoa?

SUMMARY ANSWER

Seminal plasma AEA levels were lower in men with asthenozoospermia and oligoasthenoteratozoospermia compared with normozoospermic men.

WHAT IS KNOWN ALREADY

AEA, a bioactive lipid, synthesized from membrane phospholipids may signal through cannabinoid receptors (CB1 and CB2) to regulate human sperm functions and male reproduction by modulating sperm motility, capacitation and the acrosome reaction in vitro. Local AEA levels are regulated by the synthetic and degradative enzymes, NAPE-PLD and FAAH, respectively. How the deregulation of this endogenous signalling pathway affects human sperm function(s) is not clear.

STUDY DESIGN, SIZE AND DURATION

This was a cross-sectional study of 86 men presenting at an infertility clinic for semen analysis over a period of 2 years.

PARTICIPANTS/MATERIALS, SETTING, METHODS

AEA was quantified, by ultra-high performance liquid chromatography-tandem mass spectrometry, in seminal plasma from 86 volunteers. Using qRT–PCR, CB1, CB2, NAPE-PLD and FAAH transcript levels were determined in spermatozoa from men with normozoospermia, asthenozoospermia, oligoasthenoteratozoospermia and teratozoospermia. Normal spermatozoa were exposed in vitro to methanadamide (meth-AEA) to determine its effect on sperm motility, viability and mitochondrial activity.

MAIN RESULTS AND THE ROLE OF CHANCE

Seminal plasma AEA levels (mean ± SEM) were significantly lower in men with asthenozoospermia (0.080 ± 0.01 nM; P < 0.05) or oligoasthenoteratozoospermia (0.083 ± 0.01 nM; P < 0.05) compared with normozoospermic men (0.198 ± 0.03 nM). In addition, the levels of spermatozoal CB1 mRNA were significantly decreased in men with asthenozoospermia (P < 0.001) or oligoasthenoteratozoospermia (P < 0.001) compared with normozoospermic controls. Supra-physiological levels of meth-AEA decreased sperm motility and viability, probably through CB1-mediated inhibition of mitochondrial activity.

LIMITATIONS, REASONS FOR CAUTION

The inhibitory effect of meth-AEA was only shown in vitro and may not reflect what happens in vivo.

WIDER IMPLICATIONS OF THE FINDINGS

As the regulation of the endocannabinoid system appears to be necessary for the preservation of normal sperm function and male fertility, there may be implications for the adverse reproductive consequences of marijuana use. Exocannabinoids, such as ⁹-THC, are likely to compete with endocannabinoids at the cannabinoid receptors, upsetting the finely balanced endocannabinoid signalling system. The importance of the endocannabinoid system makes it an attractive target for pharmacological interventions to control male fertility.

STUDY FUNDING/COMPETING INTEREST(S)

This work was funded in part by miscellaneous educational funds from the University Hospitals of Leicester National Health Services Trust to support the Endocannabinoid Research Laboratory of University of Leicester. The authors declare no competing interests.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/8/2058?rss=1

Recommendation and review posted by G. Smith

STUDY QUESTION

When does a difference in human intrauterine growth of singletons conceived after IVF and embryo culture in two different culture media appear?

SUMMARY ANSWER

Differences in fetal development after culture of embryos in one of two IVF media were apparent as early as the second trimester of pregnancy.

WHAT IS KNOWN ALREADY

Abnormal fetal growth patterns are a major risk factor for the development of chronic diseases in adult life. Previously, we have shown that the medium used for culturing embryos during the first few days after fertilization significantly affects the birthweight of the resulting human singletons. The exact onset of this growth difference was unknown.

STUDY DESIGN, SIZE AND DURATION

In this retrospective cohort study, all 294 singleton live births after fresh embryo transfer in the period July 2003 to December 2006 were included. These embryos originated from IVF treatments that were part of a previously described clinical trial. Embryos were allocated to culture in either Vitrolife or Cook commercially available sequential culture media.

PARTICIPANTS/MATERIALS, SETTING, METHODS

We analysed ultrasound examinations at 8 (n = 290), 12 (n = 83) and 20 weeks’ (n = 206) gestation and used first-trimester serum markers [pregnancy-associated plasma protein-A (PAPP-A) and free β-hCG]. Differences between study groups were tested by the Student's t-test, ² test or Fisher's exact test, and linear multivariable regression analysis to adjust for possible confounders (for example, parity, gestational age at the time of ultrasound and fetal gender).

MAIN RESULTS AND THE ROLE OF CHANCE

A total of 294 singleton pregnancies (Vitrolife group n_VL = 168, Cook group: n_C = 126) from 294 couples were included. At 8 weeks’ gestation, there was no difference between crown-rump length-based and ovum retrieval-based gestational age (GA) (n_VL = 163, n_C = 122, adjusted mean difference, –0.04 days, P = 0.84). A total of 83 women underwent first-trimester screening at 12 weeks’ gestation (n_VL = 45, n_C = 38). GA, nuchal translucency (multiples of median, MoM) and PAPP-A (MoM) did not differ between the study groups. Free β-hCG (MoM) ± SEM differed significantly (1.55 ± 0.19 in Vitrolife versus 1.06 ± 0.10 in Cook; P = 0.031, Student's t-test). At 20 weeks’ gestation, a more advanced GA, reflecting an increased fetal growth, was seen at ultrasound examination in the Vitrolife group (n = 115) when compared with the Cook group (n = 91). After adjustment for confounding factors, both the difference between GA based on three biparietal diameter dating formulas minus the actual (ovum retrieval based) GA (adjusted mean difference + 1.14 days (P = 0.04), +1.14 days (P = 0.04) and +1.36 days (P = 0.048)), as well as head circumference (HC) and trans-cerebellar diameter (TCD) were significantly higher in the Vitrolife group (HC_vl 177.3 mm, HC_c 175.9 mm, adjusted mean difference 1.8, P = 0.03; TCD_vl 20.5 mm, TCD_c 20.2 mm, adjusted mean difference 0.4, P = 0.008).

LIMITATIONS, REASONS FOR CAUTION

A first trimester (12 weeks) fetal screening was not yet offered routinely during the study period, therefore only 28% of women in our study participated in this elective screening programme. Although all sonographers were experienced and specially trained to perform these ultrasound examinations and were unaware of the randomization procedure, we cannot totally rule out possible intra- and inter-observer variability. Despite being indispensable in daily practice, sonographic weight formulas have a limited accuracy.

WIDER IMPLICATIONS OF THE FINDINGS

According to the fetal origins hypothesis, many adult diseases originate in utero owing to adaptations made by the fetus to the environment it encounters. This study indicates that the embryonic environment is already important for fetal development. Therefore, our study emphasizes the need to investigate fetal growth patterns after assisted reproduction technologies and long-term health outcomes of IVF children, especially in relation to the culture medium used during the first few days of preimplantation development.

TRIAL REGISTRATION NUMBER

Not applicable.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/8/2067?rss=1

Recommendation and review posted by G. Smith

STUDY QUESTION

Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage?

SUMMARY ANSWER

IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls.

WHAT IS KNOWN ALREADY

Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤12 completed weeks from last menstrual period) and late (≥13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role.

STUDY DESIGN, SIZE, DURATION

This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30).

PARTICIPANTS/MATERIALS, SETTING, METHODS

Total adherent decidual cells, CD10⁺ stromal cells, CD14⁺ macrophages and CD56⁺ uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8–10 or 12–14 weeks' gestation. In addition, CD14⁺ macrophages and CD56⁺ uNK cells were isolated from decidua from sporadic miscarriage at 8–10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14⁺ macrophages and CD56⁺ uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling.

MAIN RESULTS AND THE ROLE OF CHANCE

IL-8 was secreted by total adherent decidual cells, CD10⁺ stromal cells and CD14⁺ macrophages at both 8–10 and 12–14 weeks' gestation, with CD14⁺ cells secreting the highest levels. Both CD14⁺ and CD56⁺ cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling.

LIMITATIONS, REASONS FOR CAUTION

By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage.

WIDER IMPLICATIONS OF THE FINDINGS

Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling.

STUDY FUNDING/COMPETING INTEREST(S)

This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare.

TRIAL REGISTRATION NUMBER

Not applicable.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/8/2075?rss=1

Recommendation and review posted by G. Smith

STUDY QUESTION

How does vitrification affect oocyte viability?

SUMMARY ANSWER

Vitrification does not affect oocyte viability in oocyte donation cycles.

WHAT IS KNOWN ALREADY

Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs.

STUDY DESIGN, SIZE, DURATION

This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included.

PARTICIPANTS/MATERIALS, SETTING, METHODS

The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks.

MAIN RESULTS AND ROLE OF CHANCE

A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate (40.8 versus 33.3%) or implantation rate (21.8 versus 26.8%).

LIMITATIONS, REASONS FOR CAUTION

The oocytes were donated by healthy, young women (≤35 years) and these results cannot be extrapolated to other populations.

WIDER IMPLICATIONS OF THE FINDINGS

Outcomes obtained with vitrified oocytes are as good as with fresh oocytes and the use of vitrification can be extended to new applications, e.g. accumulation of oocytes from successive stimulations for preimplantation genetic diagnosis, for patients at risk of ovarian hyperstimulation syndrome or in patients needing to preserve their fertility.

STUDY FUNDING/COMPETING INTEREST(S)

This work was done under the auspices of the Càtedra d'Investigació en Obstetrícia i Ginecologia of the Universitat Autònoma de Barcelona.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/8/2087?rss=1

Recommendation and review posted by G. Smith

STUDY QUESTION

Does vitrification and warming of Day 3 embryos have an impact on neonatal outcome when compared with Day 3 embryos that are slow cooled and thawed, or with embryos from a fresh cycle?

SUMMARY ANSWER

The median birthweight was higher in the vitrified group versus the slow cooled or fresh embryo transfer, and the rate of low birthweight in twin babies was lower in the vitrified group.

WHAT IS KNOWN ALREADY

Vitrification has been successfully used for cryopreserving human oocytes and blastocyst-stage embryos. Most published studies looking at the neonatal outcomes after transfer of vitrified embryos refer to blastocyst-stage embryos. Information on children born after transfer of Day 3 vitrified embryos is relatively rare.

STUDY DESIGN, SIZE, DURATION

A retrospective, single-centre study of children born after Day 3 embryo transfer from fresh, slow frozen or vitrified embryos during the period January 2006 to May 2011 was conducted. Each patient contributed only one cycle per group. Children born were followed-up at 7–30 days after delivery. Outcome measures include obstetric and neonatal outcomes, which were evaluated by medical records and questionnaires sent to parents.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Patients underwent transfer of vitrified Day 3 embryos (n = 2617 transfers, Cryotop method), slow frozen Day 3 embryos (n = 4681) and fresh Day 3 embryos (n = 9194). All cycles were performed at the Shanghai Ji Ai Genetics & IVF Institute.

MAIN RESULTS AND THE ROLE OF CHANCE

Frequencies of hypertensive disorder, gestational diabetes, placenta previa and abruptio placenta were similar in all groups. Five hundred and forty five, 986 and 1914 singleton babies were born from vitrified, slow freezing and fresh transfers, and the median gestational ages were 38.7, 38.7 and 38.7 weeks, respectively. Preterm birth (32–37 weeks) occurred in 7.5, 9.2 and 7.8% of the vitrified, slow freeze and fresh groups, respectively. The median birthweight from vitrified embryos (3455.3 g) was higher than that from slow freezing (3352.3 g) and fresh (3355.8 g) transfers (P < 0.0001 for both). The rate of perinatal mortality was 0.4% for all transfer groups. Three hundred and eighty two, 734 and 1322 twin babies were born from vitrified, slow freezing and fresh transfers, respectively. There were no differences among groups in mean gestational age and in the rate of preterm birth. The median birthweight for babies born from vitrified embryos (2587.4 g) was higher than that from the slow freezing (2538.8 g) or fresh (2494.4 g) transfer groups (vitrified versus fresh: P = 0.0015; vitrified versus slow freeze: P = 0.049). The rate of low birthweight (1500–2500 g) from vitrified (30.4%) was lower than that from fresh (36.2%) transfer (P = 0.034).

LIMITATIONS, REASONS FOR CAUTION

The main limitation of this study is that the obstetric and neonatal data were obtained by questionnaires sent to the parents without checking medical records. This is, especially, problematic for reporting on congenital malformations, so birth defects were excluded from the data.

WIDER IMPLICATIONS OF THE FINDINGS

Transfer of vitrified and warmed Day 3 embryos does not seem to have an adverse effect on neonatal outcome. Children born following the transfer of vitrified embryos seem to have a higher birthweight when compared with those of fresh or slow frozen embryos.

STUDY FUNDING/COMPETING INTEREST(S)

This study received no outside funding and none of the authors has any conflict of interest.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/8/2093?rss=1

Recommendation and review posted by G. Smith