CLINICAL ASSISTED REPRODUCTION: Development of Blastocyst-Stage Embryos After Round Spermatid Injection in Patients with Complete Spermiogenesis Failure

Purpose: Our purpose was to evaluate the progression of embryos derived from round spermatid injection to the blastocyst stage and compare the results with those obtained by the use of testicular or epididymal spermatozoa.Methods: Thirty-eight patients with azoospermia enrolled in this study. In 29 patients with obstructive or nonobstructive azoospermia, spermatozoa were recovered from epididymis or testis. In the remaining nine cases with nonobstructive azoospermia, only round spermatids were found in seven, whereas in two of the patients, there were no elongated or round spermatids. Six of these cases underwent round spermatid injection.Results: Twenty-one of 29 patients with injection of spermatozoa underwent embryo transfer on day 3, and 10 pregnancies (47.6%) were obtained. In eight cycles, embryos were further cultured for delayed transfer. In six cases undergoing round spermatid injection, no transfer was performed on day 3 and extended culture with delayed embryo transfer was applied. The mean number of fertilized oocytes and mean number of embryos on day 3 and also the fertilization rate and mean number of good-quality embryos on day 3, mainly grade 1 or 2, were statistically significantly higher in the spermatozoa group than the round spermatid injection group. Compared to the spermatozoa group, the number of arrested embryos was significantly higher and the number of blastocyst-stage embryos and number of good-quality blastocysts were significantly lower in the spermatid injection group. No blastocysts developed in two spermatid cycles and embryo transfer was not possible, and in the remaining four cycles, after at least one blastocyst transfer, no pregnancies were achieved. However, in eight cycles with extended culture in the spermatozoa group, embryo transfers were achieved in all and three pregnancies, for a pregnancy rate of 37.5%, were obtained after blastocyst transfer.Conclusions: Our preliminary results showed that round spermatid injection was associated with a significantly lower fertilization and embryo development rate and a significantly higher developmental arrest rate compared with the injection of spermatozoa. Extended culture and delayed embryo transfer did not improve the clinical outcome after round spermatid injection, and these results suggested a developmental failure in embryos preventing successful implantation after round spermatid injection.     

Five years experiences with microinjection of testicular spermatozoa into oocytes in Hungary

The aim of the study was to summarize our five years experience (1996-2000) of testicular spermatozoa for intracytoplasmic sperm injection in Hungary. The influence of sperm count, maternal age, number of transferred embryos, and application of assisted hatching on outcome was investigated. Testicular spermatozoa were retrieved by microsurgical testicular sperm extraction. Samples were classified depending on the number of spermatozoa. Indication for testicular sperm extraction in conjunction with intracytoplasmic sperm injection was severe azoospermia or azoospermia combined with tubal origin infertility. Ovarian stimulation was carried out using an ultrashort protocol with GnRH agonist and gonadotrophin. Intracytoplasmic sperm injection was performed without PVP. Embryos were cultured for 48 or 72 h before embryo transfer. Indications for assisted hatching included elevated maternal age, increased zona thickness or at least two previous unsuccessful IVF cycles. Testicular spermatozoa were successfully retrieved in 218 out of 273 cases. Extreme low sperm count was found more frequently in cases of nonobstructive azoospermia. No significant differences were observed in fertilization rate (61.1% vs. 51.7%) or clinical pregnancy rate (29.0% vs. 26.7%) between patients with obstructive or nonobstructive azoospermia. Maternal age, number of transferred embryos and application of assisted hatching had a significant effect on outcome. A total of 55 clinical pregnancies were achieved, including 14 sets of twins, three sets of triplets and two sets of quadruplets. It is concluded that testicular sperm extraction is an efficient way of obtaining testicular spermatozoa, allowing not only successful fertilization by ICSI, but also freezing of testicular spermatozoa for use in subsequent cycles.     

Criteria predicting the absence of spermatozoa in the Sertoli cell-only syndrome can be used to improve success rates of sperm retrieval

In patients with non-obstructive azoospermia, testicular sperm extraction (TESE) is a method of choice to recover spermatozoa as a male therapeutic approach in intracytoplasmic sperm injection (ICSI) programmes. However, the efficacy of TESE in this indication is burdened by a frequent failure of sperm recovery, which renders useless both the invasive testicular intervention and ovarian stimulation of the patient's spouse. One of the most frequent pathological pictures characterizing complete absence of spermatozoa is germinal aplasia (Sertoli cell- only syndrome or SCOS). Two different histological patterns of SCOS have been already described during the past five decades. These two patterns can be characterized as the congenital (pure) and the secondary (mixed) forms. Both patterns, with different prognosis to retrieve spermatozoa by therapeutic testicular biopsy, are frequently confused when TESE is performed during ICSI programmes. Useful criteria to predict the absence of spermatozoa can be obtained by a definite recognition of the two typical histological patterns during the diagnostic testicular biopsy. The diagnosis of congenital or acquired SCOS can be refined by endocrine, chemical, immunohistochemical and molecular biology aids. Reduction of both sperm retrieval failure and unnecessary ovarian stimulation can be achieved by combination of these methods.     

Cystic fibrosis gene mutations and infertile men with primary testicular failure

It has been proposed that the gene responsible for cystic fibrosis, called the cystic fibrosis transmembrane conductance regulator (CFTR) gene, may play an important role in the process of spermatogenesis. A group of azoospermic men with primary testicular failure underwent CFTR mutation analysis, including assessment of the intron 8 polythymidine tract (IVS8-T tract). An association was not found between CFTR mutations or the 5T variant of the IVS8-T tract and the primary testicular failure phenotype. This finding suggests that CFTR does not play a significant role in the aetiopathogenesis of primary spermatogenic dysfunction. Therefore, the abnormal testicular histological findings in some post-pubertal men with cystic fibrosis may be a result of nutritional deficiency or testicular obstruction rather than a primary defect in spermatogenesis. In addition, the decreased sperm count in oligozoospermic men with CFTR mutations may be secondary to partial reproductive tract obstruction and not abnormal spermatogenesis. Lastly, routine screening of men with primary testicular failure for CFTR gene mutations is not warranted.     

GENETIC ANOMALIES DETECTED IN PATIENTS WITH NON-OBSTRUCTIVE AZOOSPERMIA AND OLIGOZOOSPERMIA

Genetic factors have a major importance in male infertility etiology. Numerical and structural chromosomal abnormalities seem to be frequent inoligospermia and azoospermia cases with unknown etiology. In this study, 819 patients with azoospermia (383) and oligospermia (436) who attended the infertility department between 1995–2005 were evaluated. Spermogram and basic hormone proties (FSH-testosterone) were studied two times in a one month interval from each patient, and all the cases were evaluated cytogenetically. The 47 (12%) of 383 azoospermia patients and the 20 (4%) of 436 oligospermia patients were found to have chromosomal abnormalities. The 9 (19%) of the chromosomal abnormalities found in azoospermia patients were autosomal and the 38 (80%) were gonosomal. In oligospermia cases, the 8 (40%) of the chromosomal abnormalities were autosomal and 12 (60%) were gonosomal. Cytogenetic analysis and genetic counseling would be helpful in infertile males with azoospermia and oligospermia by determining the genetic factors causing infertility and by assessing the genetic risks of the offsprigs provided by assisted reproductive techniques.     

Cytogenetic and molecular study of 370 infertile men in South India highlighting the importance of copy number variations by multiplex ligation-dependent probe amplification

Male infertility is a common and severe problem affecting 7% of population. The main objective of this study is to identify the chromosomal abnormalities, Y microdeletions in infertile men and also to access the frequency of abnormal sperm count. Based on the sperm count and viability, the infertile men were grouped as Azoospermia, Asthenospermia, Oligospermia and the remaining as Idiopathic infertility. A total of 370 infertile men and 60 normal control men were recruited. Chromosomal abnormalities were identified in 3 men (3/370). The prevalence of Y microdeletions in the infertile group is 8/370 in the Azoospermia factor (AZF) region with four AZFc deletion/duplication, two AZFa deletion, one AZF b & AZFc deletion and one case of total AZF a, AZFb & AZFc deletion. However, only five cases of Y microdeletions were identified by Multiplex PCR but an additional three cases by MLPA (Multiplex ligation-dependent probe amplification). Fluorescence in situ hybridisation also confirmed the deletions. Here, we performed MLPA post-multiplex PCR, and our study revealed good yield of the Y microdeletion identification. The partial duplications which are difficult to be identified can now be easily identified by MLPA, and hence, we recommend MLPA as the choice of investigation compared to multiplex PCR for infertile men.     

Aberrant methylation of the TDMR of the GTF2AIL promoter does not affect fertilisation rates via TESE in patients with hypospermatogenesis

Increasing evidence shows a relationship between epigenetic regulation and male infertility. The GTF2A1L gene promoter contains the DNA methylation site of a tissue-specific differentially methylated region （TDMR）. Eighty-six patients with non-obstructive azoospermia were assessed for the DNA methylation state of CpG islands in the GTF2AIL promoter using testicular genomic DNA. Based on histological criteria, 26 of the 86 patients had normal spermatogenesis （controls）, 17 had hypospermatogenesis and 26 had a Sertoli cell-only phenotype or tubular sclerosis. GTF2AIL TDMR methylation was significantly lower in testes DNA from control samples than from hypospermatogenic samples （P=0.029）. Patients with hypospermatogenesis were divided into two subgroups： high DNA methylation （HM, n=5） and low DNA methylation （LM, n= 12）. The GTF2AIL TDMR methylation rate differed significantly between the HM and LM groups （P=0.0019）, and GTF2A 1L expression was significantly higher among the LM than in the HM patients （P=0.023）. High TDMR methylation was correlated with low GTF2AIL gene expression levels. Both groups demonstrated relatively good outcomes with respect to sperm retrieval, fertilisation, pregnancy and childbirth rates. We observed that aberrant GTF2AIL gene expression was not correlated with fertilisation rates. The testicular sperm extraction （TESE） technique may be used to overcome male infertility due to aberrant TDMR methvlation.     

睾丸组织Johnsen评分对非梗阻性无精子症患者取精成功率的预测

目的探讨睾丸组织病理学Johnsen评分是否可以作为非梗阻性无精子症患者取精成功的预测因素.方法选取2008年6月至2011年6月来院就诊的非梗阻性无精子症患者513例.所有患者均行详细的病史采集、体格检查、实验室检查、睾丸活检及活检组织病理学检查等.患者随访1~3年,其主要包括患者是否进一步行睾丸取精术以及取精的结果.结果总计有399例患者接受了活检同侧的睾丸取精术,其中睾丸穿刺取精成功112例,而穿刺取精失败的患者通过进一步接受睾丸显微取精术,成功取精44例.通过分析睾丸取精成功的预测因素后发现,当Johnsen评分≥7时,非梗阻性无精子症患者睾丸取精的成功率将显著提升.结论睾丸组织病理学Johnsen评分≥7可能是睾丸取精成功的预测因素.     

Microsurgical epididymal sperm aspiration: indications,techniques and outcomes

Microsurgical epididymal sperm aspiration (MESA) refers to retrieval of sperm-containing fluid from optimal areas of the epididymis that are selected and sampled using high-power optical magnification provided by an operating microscope. Retrieved sperm are subsequently used for intracytoplasmic sperm injection (ICSI) to induce fertilization and pregnancy. MESA is considered by many experts to be the gold standard technique for sperm retrieval in men with obstructive azoospermia given its high yield of quality sperm, excellent reported fertilization and pregnancy rates, and low risk of complications. However, MESA must be performed in an operating room, requires microsurgical skills and is only useful for reproduction using ICSI. Herein we present an overview of the evaluation of candidate patients for MESA, the technical performance of the procedure and the outcomes that have been reported.     

严重少精子症及非梗阻性无精子症患者Y染色体微缺失分析

目的探讨严重少精子症及非梗阻性无精子症与Y染色体长臂微缺失之间的关系。方法该病例对照研究包括216例严重少精子症、189例非梗阻性无精子症患者及100例精液参数正常的对照。采用多重PCR对Y染色体AZFa、AZFb、AZFc及AZFd区域进行检测。玷果在严重性少精子症患者中,AZF总缺失率为10．65％（23／216）,其中以AZFc区缺失最常见,占缺失的78．26％（18／23）;在非梗阻性无精子症患者中,AZF总缺失率为13．76％（26／189）,其中也以AZFc区缺失最常见,占缺失的57．69％（15／26）;在正常对照中发现1例AZFb缺失,两病例组AZF区缺失分别与对照组相比较均具有显著差异（X^2=9．066,P=0．003;X^2=10．74,P=0．001）。结论通过对Y染色体微缺失的检查可以从基因水平寻找生精障碍的原因以及为优生优育提供可靠的遗传信息依据。