efficiency and safety of human reproductive cell tissue vitrification

Vitrification is a cryopreservation technique increasingly applied in clinical practice for cells and tissue. This review article focuses mainly on the efficiency of vitrification of human reproductive cells and tissue, by analysing the clinical results reported in the literature. The second aspect discussed is safety of vitrification procedure. Different procedures and different types of carriers can be used, and in some cases vitrification requires a direct contact between cell/tissue/carrier and liquid nitrogen; this causes concern regarding the safety of this cryopreservation technique. Although the risk of contamination during cryopreservation remains negligible, this article explains how to overcome the hypothetical risk of contamination when using different types of vitrification carriers, in order to satisfy all existing directives

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

<p>Abstract</p> <p>Background</p> <p>The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).</p> <p>Methods</p> <p>A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 μm and width: 3.28+/-0.20 μm, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.</p> <p>Results</p> <p>No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).</p> <p>Conclusions</p> <p>The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.</p

Contamination of single-straw carrier for vitrification

To the Editor: We read with interest the recent article by Criado et al. (1) (http://www.fertstert.org/article/S0015-0282(10)02987-0/abstract) regarding the assessment of contamination of a single-straw ultravitrification closed carrier (Ultravit). They observed no contamination in any microdrops of medium contained in the closed devices, where as the bacteria they used for forced contamination of liquid nitrogen (LN2) were present in 45% of open carriers’ strip (Cryotop)

Breakthroughs in the human embryology laboratory... what are the real triggers?

In his article, Gabor Vajta elegantly describes how advances in mammalian embryology slow down for a number of reasons, including a lack of innovative thinking probably due to the lim- ited resources dedicated to “research and devel- opment” in this field. The picture he paints of IVF laboratory technologies “crystallized” at 20 years ago is harsh but accurate, especially when compared with other fields such as molecular genetics or mobile phones. In this scenario, the fascinating idea of a geneticist and an embryol- ogist from the early nineties time-travelling into this decade of the new century, with the geneti- cist lost in the new technology and the embryol- ogist perfectly capable of performing their usu- al lab routine, neatly gives the idea of how time has stopped in the IVF laboratory. But, particularly in the field of human IVF, some changes have taken place in the last 20 years after the “ICSI revolution” during the early nineties, undoubtedly the major break- through in this field. And it is very interesting to observe that in some circumstances new tech- nologies or procedures were introduced not to improve the final result or to simplify the laboratory routine, but to overcome difficulties caused by strict legal requirements or for marketing reasons. A striking example of the role indirectly played by legislative limitations is in the field of oocyte cryopreservation. This technique was only considered a safe procedure – and thus worth being marketed and used as a clinical tool – after a critical mass of information regarding its effi- ciency and safety had been gathered from the “Italian experience”. In 2012 the practice com- mittee of ASRM published its guidelines for oocyte cryopreservation, stating that it should no longer be considered experimental, replacing with this document the one previously published in 2008, in which they warned against the mar- keting and clinical use of oocyte and ovarian tis- sue cryopreservation. It is interesting to observe that 30% of the papers in the references were published by Italian groups, and the majority of these articles contain data collected from 2004 to 2009, the period of an abstruse, bizarre and exceedingly strict national IVF law, which was later, in 2009, declared unconstitutional and against human rights by the Italian Constitution- al Court. This law (Law 40/04) was approved by the Ital- ian Parliament in February 2004 to regulate as- sisted reproduction technology. It infringed up- on basic human rights and the proper application of IVF technology because it mandated proce- dures that were against the best interest of the woman seeking pregnancy. The main point of controversy was the combination of a mandatory limit of three embryos for transfer, and an obligation to implant all embryos produced; cryopreservation of excess embryos was prohibited. Obviously, this decreased the chances for most women of achieving pregnancy, while at thesame increasing the number and complexity of procedures they needed to undergo and exposing some to an unacceptable risk of multiple pregnancy. This 2004 law was inspired by the desire to protect every newly produced embryo and deeply influenced by the way of think- ing of the Catholic Church. In this scenario, only the cryopreservation of gametes was allowed. This involved the female gamete despite the fact that at that time the majority of scientists and in- ternational scientific societies considered oocyte cryopreservation an experimental and unsafe technique. On 8 May 2009, the Italian Constitutional Court declared that Law 40/04 was unconstitutional. The most important theoretical point made by the Court was that it did not provide protection to embryos, since it admitted that some of them may not produce a viable fetus. Embryo protection is therefore limited by the imperative to ensure a concrete possibility to achieve a success- ful pregnancy. Owing to of this law, in Italy between 2004 and 2009 oocyte cryopreservation was used in addi- tion to conventional IVF techniques as a way to circumvent many of the regulative and ethical issues associated with embryo cryopreservation. This period saw the peak in the production of papers concerning oocyte cryopreservation by Italian groups, both basic science studies and clinical reports. At the end of this period, the critical mass of clinical data obtained mainly from the “Italian experience” and a few other groups together with advances in vitrification technologies in the last 20 years, definitively boosted human oocyte cryopreservation, by demonstrating that vitrified oocytes perform as well as their fresh counterparts. Based on these stud- ies, international scientific societies finally took away the “experimental” label from this technology, which is nowadays used mostly for oocyte donation programmes (http://www.sart.org/). Other examples of new technologies which have been developed in the last 20 years of IVF in response to the introduction of strict laws or quality assurance programmes are the use of automatic witnessing/tracking systems, and improvements in lab environment sterility conditions and/or certification of aseptic procedures. In tracking systems, time lapse microscopy (TLM) plays a crucial role. And one of the major factors in the massive increase in TLM culture systems in IVF labs in the last five years, must be the advances in computer/wireless and smartphone/tablet technology, which allow patients to see the footage of their growing embryo. So, despite the potential positive effect of TLM for embryo selection, it seems that the real trigger for the explosion of this technology in IVF units is much more related to customer service and marketing reasons

Efficacy of low-dose human chorionic gonadotropin alone to complete controlled ovarian stimulation.

OBJECTIVE: To prove that low-dose hCG alone can be clinically used to replace FSH-containing gonadotropins to complete controlled ovarian hyperstimulation (COH). DESIGN: Controlled, prospective, randomized study. SETTING: Academic center. PATIENT(S): Infertile patients who are candidates for assisted reproduction. INTERVENTION(S): Patients received [1] recombinant FSH or hMG throughout COH (group A); [2] ovarian priming with recombinant FSH/hMG followed by low-dose hCG (200 IU/day) alone (group B). MAIN OUTCOME MEASURE(S): Medication consumption; daily serum and follicular fluid (FF) measurements of LH, FSH, hCG, E2, P, T, and androstenedione (A); number and size of follicles; intracytoplasmic sperm injection (ICSI) outcome. RESULT(S): In group B: [1] duration and dose of recombinant FSH/hMG administration were reduced; [2] preovulatory serum hCG, E2, and T were higher, whereas FSH was lower; [3] FF hCG, E2, T levels, and E2/T, E2/A, and E2/P ratios were higher, whereas A was lower; [4] small but not large preovulatory follicles were reduced; [5] fertilization rates were higher; and [6] serum and FF P levels, and ICSI outcome did not differ. CONCLUSION(S): Low-dose hCG alone in the late COH stages: [1] reduced recombinant FSH/hMG consumption whereas ICSI outcome was comparable to traditional COH regimens; [2] stimulated follicle growth and maturation independent of FSH administration; [3] was associated with a reduced number of small preovulatory follicles; [4] did not cause premature luteinization; [5] resulted in a more estrogenic intrafollicular environment

EFFICIENCY OF HUMAN OOCYTE SLOW FREEZING: RESULTS FROM FIVE ASSISTED REPRODUCTION CENTRES

It has been demonstrated previously that freezing oocytes within 2 h of retrieval increases the efficiency of cryopreservation via a slow-freezing/rapid-thawing protocol with 0.3 mol/l sucrose (SF/RT 0.3). The aim of this multicentre survey was to verify this observation on a larger scale. This was a retrospective study on the clinical outcome of 510 SF/RT 0.3 cycles divided into two groups: group A, freezing oocytes within 2 h of retrieval; group B, freezing oocytes more than 2 h after retrieval. The rate of best-quality embryos was significantly higher (33.24%) in group A than in group B (16.20%, P &lt; 0.001). Pregnancy and implantation rates were 30.07% and 15.08% in group A versus 8.97% and 4.57% in group B (P &lt; 0.001). Clinical pregnancy rates per thawed and per injected oocyte in group A were 5.53% and 10.41%, versus 1.46% and 2.77% in group B (P &lt; 0.001). The overall yield from oocytes cryopreserved within 2 h of retrieval (group A) was 6.49 implantations per 100 oocytes thawed versus 1.74 for group B (P &lt; 0.001). Embryo quality, pregnancy and implantation rates, and clinical efficiency of thawing cycles were all significantly improved when cryopreservation was carried out within 2 h of oocyte retrieval

Impact of medically assisted fertility on preterm birth.

Preterm birth is a frequent problem in women who undergo treatment for infertility. Many factors appear to contribute to the occurrence of this complication. Infertile women seem to have a predisposition to giving birth preterm and to having low birthweight babies. These complications also occur in women with a history of infertility who achieve pregnancy without treatment and who have singleton pregnancies. Assisted reproduction patients treated with in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) have a disproportionately high occurrence of preterm births even with singleton pregnancies. Spontaneous preterm labour may be related to underlying medical conditions of the female partner, as its occurrence is not increased in subjects treated with ICSI (i.e. when the infertility problem is associated with male reproductive dysfunction in normal female partners). Multiple pregnancy is the factor most likely to be related to preterm birth in infertile women. The administration of drugs to induce ovulation either alone or combined with intrauterine insemination causes a significant increase in multiple pregnancies. The occurrence of higher order multiple pregnancy is also increased. Multiple pregnancy in women undergoing IVF or ICSI is related to the number of embryos transferred at the end of treatment. The transfer of more than two embryos in women under 35 is not associated with an increased chance of conception, while the occurrence of multiple pregnancy is significantly increased. Women over 40 may benefit from the transfer of more than two embryos, with fewer risks of multiple pregnancy. Single embryo transfer is increasingly considered a workable clinical option, particularly in young women. Hopefully, a more cautious approach to infertility management will reduce the occurrence of multiple pregnancy, spontaneous preterm labour and the high number of low birthweight infants born after treating these women