M.C. Magli

Paternal contribution to aneuploidy in preimplantation embryos

The association between sperm indices and the chromosomal status of preimplantation embryos was assessed in 230 couples with a female partner younger than 36 years undergoing 295 cycles of preimplantation diagnosis for aneuploidy: 105 cycles had normozoospermic samples, 134 cycles presented with oligoasthenoteratozoospermia (OAT), while the remaining cycles had spermatozoa retrieved from the seminal tract due to obstructive (29 cycles) or non-obstructive azoospermia (NOA, 27 cycles). One blastomere was biopsied from day-3 embryos and analysed for chromosomes XY, 13, 15, 16, 17, 18, 21, and 22. From the testing of 1549 embryos, the proportion of chromosomally abnormal embryos was significantly lower in normozoospermic patients (55%) than in OAT (62%, P < 0.025) and NOA patients (69%, P < 0.005). Complex abnormalities were the most frequent defect in NOA (68%), which also demonstrated the highest incidence of gonosomal aneuploidy (12%). From the re-analysis of all blastomeres in 493 non-transferred embryos, 95% of NOA embryos were chaotic mosaics. In conclusion, a severe male infertility condition could contribute to the generation of chromosomal abnormalities in the resulting embryos. This might occur especially in NOA patients in which the high incidence of chromosomal abnormalities is mainly due to mosaicism and gonosomal aneuploidy.

DNA integrity is maintained after freeze-drying of human spermatozoa

OBJECTIVE To evaluate the effects on human spermatozoa of freeze-drying, also known as lyophilization, and of cryopreservation in liquid nitrogen. DESIGN Prospective experimental study. SETTING Reproductive medicine unit and a private IVF center. PATIENT(S) Thirty healthy male donors. INTERVENTION(S) Sperm samples from 30 donors divided as two aliquots, one to be lyophilized and the other to be cryopreserved in liquid nitrogen. MAIN OUTCOME MEASURE(S) Assessment of count, motility, morphology, viability, DNA integrity, chromosomal status, and birefringence properties of lyophilized and cryopreserved human spermatozoa compared with the same parameters in the fresh sample. RESULT(S) Although sperm viability and motility were totally compromised after freeze-drying, the sperm chromatin structure was not altered in comparison with fresh samples, which demonstrated that the procedure did not affect DNA integrity. The sperm-head inner protoplasmic structures were also preserved, which was estimated by assessing the corresponding birefringence characteristics. After cryopreservation with liquid nitrogen, the motility, viability, and DNA integrity of spermatozoa were statistically significantly reduced compared with the fresh samples; the proportion of sperm cells with abnormal head birefringence increased meaningfully. CONCLUSION(S) The process of freeze-drying deeply damages cell membranes; however, unlike with liquid nitrogen preservation, it does not affect DNA integrity.

Transvaginal ovarian drilling: A new surgical treatment for improving the clinical outcome of assisted reproductive technologies in patients with polycystic ovary syndrome

OBJECTIVE To evaluate the efficacy of transvaginal ovarian drilling (TVOD) in patients with polycystic ovary syndrome (PCOS) who were undergoing IVF treatment. DESIGN Pilot study. SETTING Reproductive medicine unit. PATIENT(S) Eleven patients with PCOS undergoing treatment with assisted reproductive technology (ART). INTERVENTION(S) Selection criterion for TVOD was repeated poor performance in > or =2 previous IVF cycles. MAIN OUTCOME MEASURE(S) Controlled ovarian hyperstimulation parameters, number of eggs collected, fertilization rate, embryo cleavage rate, implantation rate, pregnancy rate compared with the cycles before TVOD. RESULT(S) In the cycle after TVOD, a significantly higher dosage of FSH was used (33.5 +/- 12 IU vs. 52.2 +/- 15 IU) to collect a higher number of oocytes in the presence of similar E2 values at the day of hCG administration. This resulted in significantly higher fertilization and cleavage rates (27% vs. 66% and 54% vs. 72%, respectively). The pregnancy and the implantation rates after TVOD were similar to those for normovulatory patients undergoing IVF for tubal factor infertility during the study period. CONCLUSION(S) Our data suggest that the TVOD is effective in improving IVF results in difficult to treat patients with PCOS, and it is less invasive and less expensive when compared with laparoscopic ovarian diathermy.

Mild ovarian stimulation with clomiphene citrate launch is a realistic option for in vitro fertilization.

OBJECTIVE To validate the use of clomiphene citrate in IVF when mild stimulation approaches are chosen to reduce patient discomfort, risk, and cost. DESIGN Prospective cohort study. SETTING Private IVF clinic. PATIENT(S) A total of 163 patients undergoing IVF and with a good prognosis (defined as ≤38 years old with normal ovarian reserve and normovulatory cycles, body mass index <29 kg/m(2), no previous assisted reproductive technology cycles, no severe endometriosis, no history of recurrent miscarriage, no endocrine/autoimmune diseases, and no surgical semen extraction). INTERVENTION(S) Mild stimulation using a fixed protocol of clomiphene citrate (100 mg/d from cycle days 3 to 7) in combination with low doses of gonadotropins (150 IU of recombinant FSH on cycle days 5, 7, and 9) and GnRH antagonist. MAIN OUTCOME MEASURE(S) The cumulative delivery rate per patient after three fresh and/or frozen embryo transfers and time to pregnancy. RESULT(S) No dropouts were observed. The cumulative delivery rate was 70%, and the mean time to pregnancy was 2.4 months. CONCLUSION(S) Mild stimulation using clomiphene citrate in combination with low doses of gonadotropins can be considered a realistic option for good-prognosis patients undergoing IVF.

A Study to Sustain the Hypothesis of the Multiple Genesis of Oligoasthenoteratospermia in Human Idiopathic Infertile Males

Abstract Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploid sperm was explored to test whether oligoasthenoteratospermia (OAT) may have a multiple origin in idiopathic infertile males. A total of 174 men (age, 35.8 ± 4.3 yr) with idiopathic infertility were studied. Seven patients had nonobstructive azoospermia, 55 had severe OAT, 30 had OAT, 27 had isolated alterations of motility, 45 had alterations of morphology and of motility, and 10 had isolated alterations of morphology. The sperm morphology was assessed with strict criteria. The percentage of aneuploid sperm was assessed with fluorescent in situ hybridization for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Relationships between sperm features, and the relationship between sperm features and aneuploidies were analyzed with multivariate regression analysis. Statistical analysis did not find any significant relationship between the percentage of typical forms and sperm concentration or between morphology and motility. On the other hand, a positive and significant relationship was found between sperm concentration and motility. The percentage of aneuploid sperm was inversely and significantly related to the percentage of typical forms but not to motility and concentration. Sperm morphology is an independent characteristic with respect to concentration and motility, whereas it showed a significant inverse relationship with respect to the percentage of aneuploid sperm. This means that idiopathic OAT may occur by means of at least two independent pathways, the first affecting concentration and/or motility and the second affecting morphology.

Reiterative changes in the Italian regulation on IVF: the effect on PGD patients’ reproductive decisions

National legislations represent one of the main factors influencing access to assisted reproduction treatment. The Italian situation in the last decade is an example of how the treatment of patients for preimplantation genetic diagnosis (PGD) was more dependent on regulators than on medical choices. This report analysed how the changes in Italian regulation affected the number of PGD referrals to this study centre, as well as their decision to opt for cross-border reproductive care (CBRC). The analysis showed that during the period in which PGD was actually not performed because of the restriction imposed by the Italian law on IVF (from 24 February 2004 to 7 May 2009) there was a significant decrease in the number of referrals asking for PGD (2.5% of total referrals) compared with the previous years (3.3%; P < 0.025) and following years when PGD was legalized (5.1%; P < 0.001). The number of couples opting for CBRC had an opposite trend, reaching a maximum when PGD was banned from Italian centres (55 couples), whereas after the readmission of PGD, only eight couples went abroad for treatment. Concomitantly, since May 2009, the proportion of couples performing a PGD cycle in this centre has constantly increased.

Reduction in sperm aneuploidy levels in severe oligoasthenoteratospermic patients after medical therapy: a preliminary report

The objective of this study was to investigate whether medical therapy can reduce sperm aneuploidy levels and improve the results of intracytoplasmic sperm injection (ICSI) in patients with severe idiopathic oligoasthenoteratospermia (OAT). Thirty-three infertile couples requiring ICSI because of severe idiopathic OAT after at least one unsuccessful ICSI cycle were considered. Semen parameters (concentration, motility and morphology), the percentage of aneuploid sperm and the results of ICSI (the number of oocytes fertilized, embryos transferred, biochemical pregnancies, clinical pregnancies and live births) were compared before and after a 3-month course of treatment with L-carnitine 1 g given twice per day+acetyl-L-carnitine 500 mg given twice per day+one 30-mg cinnoxicam tablet every 4 days. Aneuploidy was assessed using fluorescent in situ hybridisation (FISH) performed on chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The results showed that 22 of the 33 patients had a reduced frequency of aneuploid sperm and improved sperm morphology after treatment (group 1), and 11 showed no change (group 2). The numbers of biochemical pregnancies, clinical pregnancies and live births were significantly higher in group 1 than in group 2. No significant difference was found between the groups regarding the numbers of oocytes fertilized and embryos transferred. The side effects were negligible. The numbers of ICSI pregnancies and live births in severe idiopathic OAT patients improved with a course of L-carnitine, acetyl-L-carnitine and cinnoxicam.

Current regulatory arrangements for assisted conception treatment in European countries

Infertility is a condition affecting an increasing number of individuals all over the world. In recent years, this phenomenon has spread across both western countries and developing countries, thus developing the features of a pandemic. For this reason, the World Health Organization (WHO) acknowledged that infertility should be considered a disease to all intents and purposes, as it diminishes the health and wellbeing of the individuals who suffers from it. At present, the most effective means to contain the spread of infertility are essentially prevention and Assisted Reproductive Technologies (ART). With reference to the latter, although most of these techniques are routinely used in the majority of countries, they are still subject to medical, ethical and political debates. There are huge variations noted when the regulatory legislation adopted by different countries to govern infertility treatment in various countries all over the world are reviewed. In fact, it has to be recognized that ART legislation depends on a variety of factors, such as social structure, political choices, ethical issues and religious beliefs. This makes it apparently impossible to create a standard regulation for different countries, especially in case of controversial issues like gamete and embryo donation, embryo cryopreservation or surrogacy.

LH Pretreatment as a Novel Strategy for Poor Responders

Introduction. Poor response to ovarian stimulation is still a major problem in IVF. The study presents a new stimulation protocol evaluated in a suppopulation of very difficult young poor ovarian responders. Material and Methods. The study consists in two sections. The first includes data from a randomized controlled study involving forty-three young patients with a poor ovarian response in at least two previous cycles (intended as cycle cancellation or with ≤3 collected oocytes). Patients were randomized in two groups: group A (control) received FSH (400 IU/day), while group B received the new stimulation protocol consisting in a sequential association of 150 IU r-LH for 4 days followed by 400 IU r-FSH/after downregulation with daily GnRh agonist. The second includes data from the overall results in 65 patients treated with the new protocol compared to their previous performance with conventional cycles (historical control). Results. Both in the RCT and in the historical control study, LH pretreatment was able to decrease the cancellation rate, to improve the in vitro performance, and to significantly increase the live birth rates. Conclusions. LH pretreatment improved oocyte quantity and quality in young repeated poor responders selected in accordance with the Bologna criteria.

PGD for the m.14487 T>C mitochondrial DNA mutation resulted in the birth of a healthy boy

We report on the first PGD performed for the m.14487 T>C mitochondrial DNA (mtDNA) mutation in the MT-ND6 gene, associated with Leigh syndrome. The female carrier gave birth to a healthy baby boy at age 42. This case adds to the successes of PGD for mtDNA mutations.