Lodovico Parmegiani

Physiologic ICSI: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality.

OBJECTIVE To evaluate the role of hyaluronic acid (HA) for sperm selection before intracytoplasmic sperm injection (ICSI). DESIGN Three prospective studies. SETTING Private assisted reproduction center in Italy. PATIENT(S) Study 1: 20 men. Study 2: 15 men. Study 3: 206 couples treated with ICSI on a limited number of oocytes per patient (1-3) in accordance with Italian IVF law. INTERVENTION(S) Study 1: determination of sperm DNA fragmentation of HA-bound spermatozoa versus spermatozoa in polyvinylpyrrolidone (PVP). Study 2: assessment of nuclear morphology of HA-bound spermatozoa versus spermatozoa in PVP. Study 3: randomized study comparing conventional PVP-ICSI to ICSI in which the spermatozoa are selected for their capacity to bind to HA (HA-ICSI). MAIN OUTCOME MEASURE(S) Study 1: sperm DNA fragmentation rate. Study 2: sperm nucleus normalcy rate according to motile sperm organellar morphology examination criteria. Study 3: fertilization, embryo quality and development, and implantation and pregnancy. RESULT(S) Spematozoa bound to HA show a significant reduction in DNA fragmentation (study 1) and a significant improvement in nucleus normalcy (study 2) compared with spermatozoa immersed in PVP. Furthermore, injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and development (study 3). CONCLUSION(S) Hyaluronic acid may optimize ICSI outcome by favoring selection of spermatozoa without DNA fragmentation and with normal nucleus. Furthermore, HA may also be used to speed up the selection of spermatozoa with normal nucleus during intracytoplasmic morphologically selected sperm injection (IMSI).

Efficiency of hyaluronic acid (HA) sperm selection

PurposeHyaluronic Acid (HA) has a role as “physiologic selector” for spermatozoa prior to intracytoplasmic sperm injection (ICSI). The objective of this study is to analyze the results achievable by the introduction of a routine HA-ICSI programme.MethodsWe retrospectively observed 293 couples treated with HA-ICSI versus 86 couples treated with conventional PVP-ICSI (historical control group). ICSI was performed on a limited number of oocytes per patient (1–3) according to Italian IVF law at the time of the study. Main outcome measures observed were: fertilization, embryo quality, implantation and pregnancy.ResultsThis study showed that Injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and implantation.ConclusionsIf wider multi-center randomized studies will confirm these beneficial effects on ICSI outcome, HA could be considered as a routine choice for “physiologic” sperm selection prior to ICSI.

Comparison of controlled ovarian stimulation with human menopausal gonadotropin or recombinant follicle-stimulating hormone

OBJECTIVE To carefully examine the features of controlled ovarian stimulation performed with recombinant FSH-alpha or hMG. DESIGN Controlled, prospective, randomized comparison of fixed gonadotropin regimens. SETTING Academic research institution. PATIENT(S) Fifty infertile patients who were candidates for IUI. INTERVENTION(S) Patients were randomized to receive a fixed regimen of recombinant FSH-alpha (150 IU/day, 25 patients) or hMG (150 IU/day, 25 patients), after GnRH-agonist suppression (long regimen). MAIN OUTCOME MEASURES Daily measurements of serum LH, immunoreactive FSH, hCG, E(2), P, and T. Transvaginal pelvic ultrasound every 2 days. Pregnancy and abortion rates. Cost of medications. Two recombinant FSH-alpha-treated patients did not respond. Despite matched daily FSH dose, duration of treatment (hMG 10.8 +/- 0.4 vs. recombinant FSH-alpha 12.4 +/- 0.5 days), gonadotropin dose (21.7 +/- 0.8 vs. 25.3 +/- 1.3 ampoules), gonadotropin cost (288 +/- 10 vs. 1,299 +/- 66 /cycle), serum P levels, and small preovulatory follicle number were significantly lower, and LH, hCG, immunoreactive FSH levels, and larger follicles on day 8 were significantly higher in hMG-treated patients. The pregnancy, abortion, and twin pregnancy rates did not differ. CONCLUSION The hMG administration was associated with: [1]. increased serum LH activity and immunoreactive FSH levels during treatment; [2]. reduced signs of premature luteinization; [3]. differential modulation of folliculogenesis; [4]. lower treatment duration, gonadotropin dose, and cost; and [5]. clinical outcome comparable to recombinant FSH-alpha.

Intracytoplasmic sperm injection pregnancy after low-dose human chorionic gonadotropin alone to support ovarian folliculogenesis

OBJECTIVE To prove that several days of low-dose hCG alone can be used to stimulate folliculogenesis, complete FSH-initiated follicle/oocyte maturation, and achieve pregnancy in assisted reproduction technology. DESIGN Case report. SETTING Reproductive endocrinology center at an academic institution. PATIENT(S) A 35-year-old female patient and her partner with male-related infertility. INTERVENTION(S) After an 8-day priming with hMG (225 IU/d), we administered low-dose hCG (200 IU/d) alone for 5 days in one GnRH-agonist suppressed patient until proper follicle development was obtained and intracytoplasmic sperm injection was performed. MAIN OUTCOME MEASURE(S) Daily serum levels of LH, FSH, hCG, E(2), P, and T; measurements of follicle number and size; oocytes retrieved and fertilized; pregnancy. RESULT(S) Although FSH levels rapidly declined after hMG discontinuation, E(2) and large follicles increased during hCG-only administration. Several good quality oocytes were retrieved and fertilized by intracytoplasmic sperm injection; three embryos were transferred and a twin pregnancy ensued. CONCLUSION(S) Replacement of FSH with low-dose hCG for several days in the late ovulation induction stages of assisted reproduction technology resulted in: [1] continued growth of large ovarian follicles and E(2); [2] an optimal preovulatory follicle pattern consisting of many large and few medium and small follicles; and [3] reproductively competent oocytes and pregnancy.

Ultra-violet sterilization of liquid nitrogen prior to vitrification

Sir, Regarding the biosafety of direct exposure of tissue/cells to liquid nitrogen (LN2) during the vitrification procedure when using ‘open carriers’, debated by Wang (2009) and Isachenko et al. (2009) in the July 2009 issue of this journal, we would like to disagree. We strongly dispute the proposed inefficacy of ultra-violet (UV) treatment of LN2 to guarantee the absence of contamination by viruses of biological material. UV disinfection is a well-accepted technology for the inactivation of bacterial and protozoan pathogens. Until recently, UV was also considered a viable technology for disinfection of viruses (Linden et al., 2007). At UV doses typically used in liquid disinfection (the UV dose is defined as the smallest amount of UV radiation able to guarantee the complete death of a given pathogen), UV is very effective against almost all known pathogenic viruses, with the single exception of adenoviruses (Gerba et al., 2002). However, according to US Environmental Protection Agency regulations (2006), the inactivation of adenoviruses can be achieved with a UV dose of 200 000 mWs/ cm. Hence, an adequate amount of UV radiation de-activates the growth of all kinds of micro-organisms, from viruses like Hepatitis (which require an 8000 UV dose) to fungi like Aspergillus niger (330 000 UV dose) (Srikanth, 1995). The use of UV radiation to sterilize water or other liquids is well known; however, this method is difficult to apply to LN2, which evaporates rapidly. Furthermore, with some liquids, the UV radiation may be absorbed before it can reach the micro-organism to be de-activated; fortunately however, LN2 is largely transparent to the radiations from the most common commercial UV sources. Recently, we demonstrated that direct UV sterilization is applicable to LN2; our study showed that decontamination of a small volume of LN2 from bacteria and fungi like Aspergillus [the most UV-resistant micro-organism ever observed in LN2 (Bielanski et al., 2003; Morris, 2005)] via UV irradiation is feasible and straightforward (Parmegiani et al., 2009). Our method for sterilizing LN2 is based on emitting the minimum dose of UV radiation necessary to kill micro-organisms that can survive at the boiling point of nitrogen (2195.82 8C) and which is irradiated in a temperature-controlled regimen, within a short time interval, before the LN2 completely evaporates. With our method, sterile LN2 can be easily obtained for any use and, in particular, for a safe vitrification procedure. Therefore, in the case of vitrification by ‘open carriers’, after cooling in UV-sterilized LN2, biological material could be enclosed in a sterile pre-cooled device for hermetical isolation at storage as suggested by Wang (2009) and/or stored in the vapour phase of LN2 (Cobo et al., 2007; Eum et al., 2009). Since some closed systems for vitrification also involve direct contact between the cells and LN2 during the warming procedure, UV sterilization of LN2 may be appropriate in these cases. In our opinion, the fact that LN2 can be quickly and safely sterilized could encourage the clinical application of human cell/tissue vitrification, both with ‘open carriers’ and with closed systems.

Blastocyst formation, pregnancy, and birth derived from human oocytes cryopreserved for 5 years

OBJECTIVE To report a live birth after the transfer of a single blastocyst derived from a human oocyte cryopreserved for 5 years. DESIGN Case report. SETTING Private assisted reproduction center. PATIENT(S) A 39-year-old woman with tubal infertility and her partner with male-related infertility. INTERVENTION(S) Oocyte cryopreservation (in 1.5 mol/L 1,2-propanediol and 0.3 mol/L sucrose by slow freezing-rapid thawing protocol) when the patient was 34 years old. Oocyte thawing after 5 years of cryostorage. Insemination by intracytoplasmic sperm injection of the three best surviving oocytes, according to the Italian law regulating assisted reproductive technology. MAIN OUTCOME MEASURE(S) Oocyte survival after thawing. Fertilization, cleavage, and embryo development into blastocyst stage. RESULT(S) Four of six mature (metaphase II) frozen oocytes survived after thawing. Three of them were injected: these three oocytes were fertilized, and one developed into a five-cell embryo on day 3. This embryo developed into blastocyst and was transferred on day 6. A healthy female neonate weighing 3,410 g was born. CONCLUSION(S) After 5 years of storage in liquid nitrogen, cryopreserved oocytes with a slow cooling-rapid thawing protocol can develop in vitro to blastocyst stage and produce a live birth.

Rapid warming increases survival of slow-frozen sibling oocytes: a step towards a single warming procedure irrespective of the freezing protocol?

Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. The aim of this study was to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification. This was a prospective study on 216 sibling oocytes randomized for either conventional rapid thawing or rapid warming with vitrification warming solution. The primary endpoint was morphological assessment of survival at 2h. Surviving oocytes were divided into two subgroups: (i) parthenogenetically activated; and (ii) fixed and observed for spindle/chromosome configuration. Secondary endpoints were parthenogenetic development and spindle/metaphase configuration. Survival rate with rapid warming was higher (92/102, 90.2%) than with rapid thawing (85/114, 74.6%; P=0.005), and after 3d of culture the rapidly warmed parthenotes had more blastomeres compared with those rapidly thawed (P=0.042). Meiotic spindle and chromosomal configuration were not significantly influenced by rapid warming or rapid thawing. The finding of this study allows IVF centres to increase the efficiency of oocyte slow freezing, enabling survival rates comparable to vitrification protocols, and potentially to optimize costs by using the same warming protocol for both slow-frozen and vitrified reproductive cells.

Birth of a baby conceived from frozen oocytes of a 40-year-old woman

The potential, limits and safety of oocyte freezing are still being explored. Female age may play a relevant role in treatment outcome. The present study is the first report of the birth and normal development of a baby conceived from frozen oocytes of a 40-year-old woman. IVF was carried out in an infertile 40-year-old woman, and seven metaphase II (MII) oocytes were obtained after ovarian stimulation. Three fresh oocytes were inseminated by intracytoplasmic sperm injection (ICSI), according to Italian law. Two embryos were transferred, but pregnancy did not occur. The four remaining MII oocytes were frozen (by slow freezing protocol) and ICSI was performed in the two oocytes surviving after thawing. Two embryos were obtained on day 2. Both embryos were transferred, resulting in a singleton pregnancy, and a healthy male baby was born. So far, the child (now 3 years old) has scored normally according to the WHO Child Growth Standards. The Denver Developmental Screening Test for psychomotor development was normal. This report demonstrates that conception and pregnancy from cryopreserved oocytes belonging to women up to 40 years of age is possible, and can yield normal children. This finding has implications for women who want to preserve their reproductive potential.

New Advances in Intracytoplasmic Sperm Injection (ICSI)

In these last twenty years, intracytoplasmic sperm injection (ICSI) has efficiently permitted the treatment of male factor infertility (Van Steirteghem et al., 1993); the direct injection of spermatozoa into ooplasm has allowed the embryologist to overcome low sperm motility, poor sperm-Zona Pellucida (ZP) binding, and defective acrosome reaction. Although ICSI has been successfully applied worldwide for several years, nevertheless we have no real knowledge regarding the hypothetical long term side effects on ICSI adults. In fact, some doubts about the safety of this technique can arise (Oehninger, 2011) due to the fact that with ICSI some check points of natural fertilization are bypassed and that some steps differ considerably from the physiological process; For instance, the introduction of the sperm tail into the ooplasm may cause sperm nuclear decondensation problems (Dozortsev et al., 1995; Markoulaki et al., 2007). It should be considered that ICSI may increase the risk of injecting spermatozoa with genetic or functional anomalies (Sakkas et al., 1997; Bonduelle et al., 2002; Marchesi & Feng, 2007; Schatten & Sun, 2009; Heytens et al., 2009; Navarro-Costa et al., 2010). For these reasons and to minimize any risk related to ICSI, any new advance in this procedure which can help the operator to restore some of the basic physiological checkpoints and to simulate the natural fertilization process should be welcome (Parmegiani et al., 2010a).

Ultraviolet radiation dose

With regard to the response of Isachenko (2011) to our letter ‘Efficacy of ultraviolet sterilization of liquid nitrogen’, we would like to point out that in the studies cited by Professor Isachenko the ultraviolet (UV) dose administered was too low for the complete inactivation of the microorganisms. Furthermore, the extremely radiationresistant bacterium Deinococcus radiodurans is inactivated (> 4 log) by administering 400,000 lWs/cm (Krisko and Radman, 2010). Hence, an adequate amount of UV radiation de-activates the growth of all kinds of microorganisms. For this reason, we specifically designed a device for UV-LN2 sterilization which ensures and certifies a minimum UV dose of 660,000 lWs/cm per each sterilization cycle (international patent pending, application no. PCT/IB2009/007801