Purpose: To review the Testicular Sperm Extraction (TESE) with and without microscopy on patients with Klinefelter Syndrome (KS).

Method: A literature search was conducted for studies comparing TESE and mTESE efficacy for men with KS. The efficacy was measured by using the SRR and PR following Intracytoplasmic Sperm Injection (ICSI). The studies used were divided into two groups: large studies with 40 or more patients, and small studies with fewer than 40 patients.

Results: Our review results demonstrate among 1,070 KS patients treated by ICSI the SRR was 46.3% and PR was 21% with mTESE and 45.6% SRR and 44.4% PR with TESE. The SRR was 9.5% higher in large studies but PR was higher for small studies by 7.8%. Higher pregnancy rates demonstrated by TESE although the sperm retrieval rate was similar in both techniques. Among 1070 patients with KS, treated by ICSI the SRR was 46.3% and PR was 21% with mTESE and 45.6% SRR and 44.4% PR with TESE.

Conclusion: mTESE seems to have no advantages in SRR and PR as compared to standard TESE. Our results and recent meta-regression analysis publication findings of similar SRR with TESE and mTESE and similar or even better PR by TESE merits further investigation by a prospective randomized study. Parameters predicting sperm retrieval in KS patients are missing. This review’s findings of similar SRR with TESE and mTESE and similar or even better PR by TESE merits further investigation by a prospective randomized study.

Introduction

The majority of males with Klinefelter Syndrome (KS) are azoospermic and have historically been considered sterile. Today, Testicular Sperm Extraction (TESE) and Intracytoplasmic Sperm Injection (ICSI) enable 37% of KS couples to have a child [1]. Despite this observed improvement, solid parameters to identify patients with KS who have fertilization potential and a chance of fatherhood are still lacking. Investigators have attempted to correlate serum testosterone, FSH and testicular volume with positive sperm retrieval outcomes. Thus far, the results have varied widely among the different studies conducted.

FSH level appears to be the most predictive variable for sperm recovery via TESE. In 2015 Guler et al. demonstrated this phenomenon in their study where they analyzed the impact of testicular histopathology on ICSI from patients with Non Obstructive Azoospermia (NOA). They classified the histopathologies into categories as follows: normal spermatogenesis, hypospermatogenesis, maturation arrest, Sertoli cells only, and peritubular hyalinization/ tubular atrophy. It was observed that higher FSH was associated with lower PR in the “maturation arrest” group [2]. Sperm Retrieval Rates (SRR) and Fertilization Rates (FR) were found to be significantly different among all testicular histological groups of NOA. FSH found to be the best predictor of a successful TESE and testicular histology significantly influenced SRR and FRs but not PR and LBR in NOA [2]. More favorable SRR after mTESE has been reported, especially in histological patterns with focal spermatogenesis like in Sertoli cell only syndrome. In patients with uniform maturation arrest the outcome of mTESE was less favorable [3]. However, retrieval of motile spermatozoa may be sufficient to disregard predictive factors and hope of ICSI fertilization and pregnancy [4].

In contrast, a recent review’s meta-regression analysis showed that age, testis volume, FSH, LH and testosterone levels did not affect the final SRR. They also reported that there was no difference between unilateral and bilateral sperm extractions [5]. Other studies looked at the effect of early hormonal therapy on sperm retrieval rates in KS patients. Once again, the results varied between different studies. Preoperative testosterone levels that were close to or within normal range were associated with a higher predicted chance of sperm extraction. This held true both for untreated patients as well as those treated with hormonal therapies such as aromatase inhibitors, clomiphene citrate or hCG [6-10]. Out of all variables investigated, most studies agreed that age is the best predictive factor for SRR in patients with KS. Higher SRR is seen in men less than 30 years of age [7,11]. Accordingly, hormonal treatment should be considered, if indicated at all, for patients over 30 years, before opting for surgical management.

There are few randomized clinical trials comparing mTESE with conventional TESE in NOA. Conducting more of these trials will help to elucidate what factors could potentially be predictive of chances of obtaining spermatozoa in these patients. More data about testicular histology patterns, FSH levels, testicular volume and method of TESE need to be investigated. In particular, the use of microscopy to magnify the operative field has been expected to significantly aid the extraction process. Studies are needed to either validate or disprove these theories, and to explore whether or not mTESE also helps to decrease complications. The standard TESE has been associated with complications including immune system reactions within the seminal tubes and blood, as well as testis hematomas during the procedure.

Methods

The aim of this review was to collect and analyze the available data from studies comparing the use of TESE versus mTESE in non-mosaic KS patient populations. A review of the literature was performed by searching Medline, PubMed, Embase and Cochrane library for articles. The following key words were used: mTESE, TESE, ICSI and PR in patients with non-mosaic Klinefelter Syndrome. Only studies with more than 9 patients, clearly reported methodology and results on PR and/or SRR were included in this review. The collected data and papers are presented in the table 1 and 2. Statistical analysis of the data was done using a two-tailed hypothesis, with a significance level set at 0.05.

Results

Information on fertility outcome after ICSI treatment of patients with KS was available in 29 studies. In 6 studies, the methodology and results were not clear and these were excluded from the review. Fourteen articles reported both SRR and PR, and 8 articles reported only the SRR. The articles that did not report PR are designated with asterisk in table 1 and were not included in the PR calculation (Tables 1 and 2).

The differences in sperm retrieval rate and pregnancy rate between mTESE and TESE techniques are presented in table 1. Collectively, 462 patients were treated with mTESE and 608 with TESE. The SRR was 46.3% and PR was 21% with mTESE compared to 45.6% SRR and 44.4% PR with TESE. The articles were further divided into groups according to large cohort sizes of 40 patients or more and small cohort sizes of less than 40. The SRR was 9.5% higher in large studies but PR was higher for small studies by 7.8%. The overall number of patients was much lower in small studies by a difference of 29.5% for SRR and 21% for PR as compared to the overall number of patients in the large studies.

Discussion

SRR was similar in both sperm retrieval techniques, however, higher PRs were found in TESE compared to mTESE couples (Table 1). Our results are consistent with the meta-regression analysis by Corona et al. reporting 44% SRR with TESE and 43% with mTESE. Among 1,248 NOA patients, treated by ICSI the PR and LBR were 43% for both techniques and results were independent of any clinical or biochemical parameters tested [5]. In our view, during the last 15 years, factors that may have influenced the above results are: a) the introduction and use of new culture media in the market, b) manufacturers failure to state the detailed ingredients of the culture media on the label, important for quality control, c) limited experience and lack of training with sperm retrieval techniques, d) the increased number of IVF laboratories and the rare incidence of KS patients permits few patients to be treated in each center, e) the transition period of sperm retrievals being performed initially only by urologists, and later by both gynecologists and urologists.

Table 2 was created to assess the differences in outcomes between small versus large studies, and TESE vs mTESE. The large studies showed a 7% higher SRR, yet an 8.8% lower PR in comparison to the smaller studies. This demonstrates that a higher sperm count after completion of harvesting is not the key to successfully achieving a pregnancy. Although it has been claimed that with the use of mTESE, the chances of picking up sperm is higher, our analysis of the literature indicates that this is not true. When comparing all studies, TESE and mTESE had a statistically insignificant difference in SRR, however the PR was much higher with TESE. An important question to be answered, then, is why mTESE yields a much lower PR? It is possible that this difference can be explained by the techniques and protocols used.

Polarized light microscopy has been used to visualize sperm since 1937 and spindle dynamics from the 1950s-1970s, verifying that the technique is compatible with live cells. Microscopy remains the primary tool enabling the morphological assessment of gametes and embryos, for IVF laboratories [12]. Concern has been raised about the potential detrimental effects during handling and analysis, during which time they are also exposed to considerable amounts of light. Studies indicate that short wavelength visible light exposure, primarily from an incandescent microscope light bulb, can impair embryo development in rodent species [13]. Visible light exposure on mouse and rabbit gametes reportedly resulted in decreased cell division and increased DNA fragmentation [14,15]. Collection of 1-cell hamster embryos using a light microscope or with fluorescent light (370-760 nm) impaired subsequent development, even when the fluorescence was used for as little as three minutes [16]. Similar results were found after exposure of hamster and mouse zygotes to fluorescent light [17]. This same phenomenon could potentially be explained these observations about mTESE.

During microscopy increased local temperature of the area in focus, has been a concern since prolonged exposure might affect gamete and embryo development [18,19] However, the modern microscopes used in the clinical setting for real-time imaging in IVF laboratories, have undergone numerous modifications to ensure the safety of the gametes and embryos [20]. The microscopes used for mTESE are usually the same as those used in neurosurgery without any calibration for the spermatozoa. Using polarized light microscopy for testicular tissue dissection and sperm extraction should be calibrated appropriately and validated for the gametes safety [12]. Additionally, the sperm identification and collection procedure should be completed quickly to reduce the time of tissue exposure to room temperature and microscope light. Exposure time and room conditions are another important detail that is not typically reported in the studies currently published.

The outcome of mTESE versus TESE in NOA was also investigated by a systematic review published in 2014. Seven out of 62 identified studies were included in the final analysis. SRR was significantly higher in the mTESE group (42.9-63%) in comparison with TESE (16.7-45%) [3]. In another retrospective cohort study among 714 men with NOA, 40.5% had successful sperm retrieval at their first TESE. In total, 261 couples had 444 ICSI cycles and 48 frozen embryo transfer cycles, leading to 129 pregnancies and 96 live birth deliveries. The expected cumulative delivery rates after six ICSI cycles were 78%. Authors indicated the intrinsic limitations related to the retrospective cohort study design [1]. Fewer complications were observed on ultrasound examination after mTESE procedure [3].

Conclusion

Our review results demonstrate among 1,070 KS patients treated by ICSI the SRR was 46.3% and PR was 21% with mTESE and 45.6% SRR and 44.4% PR with TESE. Parameters predicting sperm retrieval in KS patients are missing. This review’s findings of similar SRR with TESE and mTESE and similar or even better PR by TESE merits further investigation by a prospective randomized study.

1. Vloeberghs V, Verheyen G, Haentjens P, Goossens A, Polyzos NP, Tournaye H. How successful is TESE-ICSI in couples with non-obstructive azoospermia.  Hum Reprod. 2015; 30: 1790-1796. https://goo.gl/qZEG4n
2. Guler I, Erdem M, Erdem A, Demirdag E, Tunc L, Bozkurt N, et al. Impact of testicular histopathology as a predictor of sperm retrieval and pregnancy outcome in patients with non-obstructive azoospermia: correlation with clinical and hormonal factors. Andrologia. 2016; 48: 765-773. https://goo.gl/aYnkNX
3. Deruyver Y, Vanderschueren D, Van der Aa F. Outcome of microdissection TESE compared with conventional TESE in non-obstructive azoospermia: A systematic review. Andrology. 2014; 2: 20-22. https://goo.gl/h9APdT
4. Salehi P, Derakhshan Horeh M, Nadeali Z, Hosseinzadeh M, Sadeghi E, Izadpanahi MH, et al. Factors influencing sperm retrieval following testicular sperm extraction in non-obstructive azoospermia patients. Clin Exp Reprod Med. 2017; 44: 22-27. https://goo.gl/74N862
5. Corona G, Pizzocaro A, Lanfranco F, Garolla A, Pelliccione F, Vignozzi L, et al. Sperm recovery and ICSI outcomes in Klinefelter syndrome: A systematic review and meta-analysis.  Hum Reprod Update. 2017; 23: 265-275. https://goo.gl/rXE2eP
6. Koichi Kyono , Hirofumi Uto, Yukiko Nakajo, Shima Kumagai, Yasuhisa Araki, Satoru Kanto. Seven pregnancies and deliveries from non-mosaic Klinefelter syndrome patients using fresh and frozen testicular sperm.  J Assist Reprod Genet. 2007; 24: 47-51. https://goo.gl/3p8Exs
7. Okada H, Goda K, Yamamoto Y, Sofikitis N, Miyagawa I, Mio Y, et al. Age as a limiting factor for successful sperm retrieval in patients with nonmosaic Klinefelter’s syndrome.  Fertil Steril. 2005; 84: 1662-1664. https://goo.gl/2psQhZ
8. Madgar I, Dor J, Weissenberg R, Raviv G, Menashe Y, Levron J. Prognostic value of the clinical and laboratory evaluation in patients with nonmosaic Klinefelter syndrome who are receiving assisted reproductive therapy. Fertil Steril. 2002; 77: 1167-1169. https://goo.gl/cSg3Fo
9. Ramasamy R, Ricci JA, Palermo GD, Gosden LV, Rosenwaks Z, Schlegel PN. Successful Fertility Treatment for Klinefelter’s Syndrome. J Urol. 2009; 182: 1108-1113. https://goo.gl/p9fGFL
10. Mehta A, Paduch DA. Klinefelter syndrome: An argument for early aggressive hormonal and fertility management. Fertil Steril. 2012; 98: 274-283. https://goo.gl/6BzixT
11. Bakircioglu ME, Ulug U, Erden HF, Tosun S, Bayram A, Ciray N. Klinefelter syndrome: Does it confer a bad prognosis in treatment of non-obstructive azoospermia. Fertil Steril. 2011; 95: 1696-1699. https://goo.gl/Sa2ujo
12. Jasensky J, Swain JE. Peering Beneath the Surface: Novel Imaging Techniques to Noninvasively Select Gametes and Embryos for ART. Biol Reprod. 2013; 89: 105. https://goo.gl/oNysVJ
13. Ottosen LD, Hindkjaer J, Ingerslev J. Light exposure of the ovum and preimplantation embryo during ART procedures.  J Assist Reprod Genet. 2007; 24: 99-103. https://goo.gl/F5FVxD
14. Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM, Okano A. Assessment of DNA damage in individual hamster embryos by comet assay. Mol Reprod Dev. 1999; 54: 1-7. https://goo.gl/QZ15LS
15. Barlow P, Puissant F, Van der Zwalmen P, Vandromme J, Trigaux P, Leroy F. In vitro fertilization, development, and implantation after exposure of mature mouse oocytes to visible light. Mol Reprod Dev. 1992; 33: 297-302. https://goo.gl/ZQtTtC
16. Umaoka Y, Noda Y, Nakayama T, Narimoto K, Mori T, Iritani A. Effect of visual light on in vitro embryonic development in the hamster. Theriogenology. 1992; 38: 1043-1054. https://goo.gl/kbRJP8
17. Takenaka M, Horiuchi T, Yanagimachi R. Effects of light on development of mammalian zygotes. Proc Natl Acad Sci U S A. 2007; 104: 14289-14293. https://goo.gl/4Ny2nV
18. Gomes CM, Silva CA, Acevedo N, Baracat E, Serafini P, Smith GD. Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment. Fertil Steril. 2008; 90: 1396-1404. https://goo.gl/iDXLpG
19. Sereni E, Sciajno R, Fava L, Coticchio G, Bonu MA, Borini A. A PolScope evaluation of meiotic spindle dynamics in frozen-thawed oocytes. Reprod Biomed Online. 2009; 19: 191-197. https://goo.gl/ZrNxdq
20. Silva CP, Kommineni K, Oldenbourg R, Keefe DL. The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. Fertil Steril. 1999; 71: 719-721. https://goo.gl/jY9weU
21. T Ishikawa, S Mizuta, K Yamaguchi, Y Takaya, R Nishiyama, K Kitaya, et al. Clinical outcomes of microdissection Testicular Sperm Extraction (micro TESE) and Intracytoplasmic Sperm Injection (ICSI) in patients with Klinefelter syndrome. Fertil Steril. 2016; 106: 298. https://goo.gl/qrmYM5
22. Hakan Ozveri,  Furkan Kayabasoglu, Cem Demirel, Ersan Donmez, Outcomes of micro-dissection TESE in patients with non-mosaic klinefelter’s syndrome without hormonal treatment.  Int J Fertil Steril. 2015; 8: 421-428. https://goo.gl/zXZpmr
23. Sabbaghian M, Modarresi T, Hosseinifar H, Hosseini J, Farrahi F, Dadkhah F, et al. Comparison of sperm retrieval and intracytoplasmic sperm injection outcome in patients with and without Klinefelter syndrome.  Urology. 2014; 83: 107-110. https://goo.gl/xRB26m
24. Ando M, Yamaguchi K, Chiba K, Miyake H, Fujisawa M. Outcome of microdissection testicular sperm extraction in azoospermic patients with Klinefelter syndrome and other sex-chromosomal anomalies. Syst Biol Reprod Med. 2013; 59: 210-213. https://goo.gl/FayySV
25. Greco E, Scarselli F, Minasi MG, Casciani V, Zavaglia D, Dente D, et al. Birth of 16 healthy children after ICSI in cases of nonmosaic Klinefelter syndrome. Hum Reprod. 2013; 28: 1155-1160. https://goo.gl/ky1XrU
26. Okada H, Goda K, Muto S, Maruyama O, Koshida M, Horie S. Four pregnancies in nonmosaic Klinefelter’s syndrome using cryopreserved-thawed testicular spermatozoa.  Fertil Steril. 2005; 84: 1508. https://goo.gl/he32wv
27. Schiff JD, Palermo GD, Veeck LL, Goldstein M, Rosenwaks Z, Schlegel PN. Success of testicular sperm injection and intracytoplasmic sperm injection in men with Klinefelter syndrome. J Clin Endocrinol Metab. 2005; 90: 6263-6267. https://goo.gl/H3y6gb
28. Vicdan K, Akarsu C, Sozen E, Buluç B, Vicdan A, Yılmaz Y, et al. Outcome of intracytoplasmic sperm injection using fresh and cryopreserved-thawed testıcular spermatozoa in 83 azoospermic men with Klinefelter syndrome. J Obstet Gynaecol Res. 2016; 42: 1558-1566. https://goo.gl/bHakus
29. Cetinkaya M, Onem K, Zorba OU, Ozkara H, Alici B. Evaluation of microdissection testicular sperm extraction results in patients with non-obstructive azoospermia: Independent predictive factors and best cutoff values for sperm retrieval.  Urol J. 2015; 12: 2436-2443. https://goo.gl/2csTuP
30. Madureira C, Cunha M, Sousa M, Neto AP, Pinho MJ, Viana P, et al. Treatment by testicular sperm extraction and intracytoplasmic sperm injection of 65 azoospermic patients with non-mosaic Klinefelter syndrome with birth of 17 healthy children. Andrology. 2014; 2: 623-631. https://goo.gl/KqHS5A
31. Selice R, Di Mambro A, Garolla A, Ficarra V, Iafrate M, Ferlin A, et al. Spermatogenesis in Klinefelter syndrome. J Endocrinol Invest. 2010; 33: 789-793. https://goo.gl/S7nYec
32. Yarali H, Polat M, Bozdag G, Gunel M, Alpas I, Esinler I, et al. TESE-ICSI in patients with non-mosaic Klinefelter syndrome: a comparative study.  Reprod Biomed Online. 2009; 18: 756-760. https://goo.gl/Wh2cGH
33. Vernaeve V, Staessen C, Verheyen G, Van Steirteghem A, Devroey P, Tournaye H. Can biological or clinical parameters predict testicular sperm recovery in 47, XXY Klinefelter’s syndrome patients. Hum Reprod. 2004; 19: 1135-1139. https://goo.gl/L5dL3s
34. Seo JT, Park YS, Lee JS. Klinefelter Successful testicular sperm extraction in Korean syndrome. Urology. 2004; 64: 1208-1211. https://goo.gl/T41BRF
35. Westlander G, Ekerhovd E, Bergh C.  Low levels of serum inhibin B do not exclude successful sperm recovery in men with nonmosaic Klinefelter syndrome.  Fertil Steril. 2003; 79: 1680-1682. https://goo.gl/kgERHj
36. Friedler S, Raziel A, Strassburger D, Schachter M, Bern O, Ron El R. Outcome of ICSI using fresh and cryopreserved-thawed testicular spermatozoa in patients with non-mosaic Klinefelter’s syndrome. Hum Reprod. 2001; 16: 2616-2620. https://goo.gl/gZSNaU
37. Levron J, Aviram Goldring A, Madgar I, Raviv G, Barkai G, Dor J. Sperm chromosome analysis and outcome of IVF in patients with non-mosaic Klinefelter’s syndrome. Fertil Steril. 2000; 74: 925-929. https://goo.gl/h64Rrr
38. Tournaye H, Staessen C, Liebaers I, Van Assche E, Devroey P, Bonduelle M, et al. Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum Reprod. 1996; 11: 1644-1649. https://goo.gl/uX23J5