How To Treat Anospermia

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1 I IOKTH IA KAI YNTA H - EK OTH : E HNIKH AN PO O IKH ETAIPEIA EÓ ÔÎÚÈÓÔÏÔÁÈÎfi TÌ Ì NÔÛÔÎÔÌÂ Ô E ENA BENIZE OY Ï Ù E. BÂÓÈ ÏÔ 2 - Aı Ó TËÏ Fax : Copyright - EÏÏËÓÈÎ AÓ ÚÔÏÔÁÈÎ EÙ ÈÚ ÓÙ. T ÙÏÔ - AÓ Ú ISSN EK OTH :.A.A ÌfiappleÔ ÏÔ TÚÈÌËÓÈ Î ÔÛË. EÎ ÂÙ È Û 2000 ÓÙ Ù apple. EappleÈÌ ÏÂÈ ÂÎÙ appleˆûë, ÛÂÏÈ ÔappleÔ ËÛË: MEDLINE ANIR is published quarterly as the official Journal of the Hellenic Society of Andrology Copyright : Hellenic Society of Andrology Short title: ANIR ISSN C o r r e s p o n d a n c e : D.A. Adamopoulos, MD, Endocrine Department Elena Venizelou Hospital, 2, E. Venizelou Square, Athens, Greece Tel : (01) , (01) , Fax : (01) hel-soc-andro@ath.forthnet.gr ANHP ANIR E I HMH EK O H TH E HNIKH AN PO O IKH ETAIPEIA OFFICIAL JOURNAL OF THE HELLENIC SOCIETY OF ANDROLOGY TÔ appleâúèô ÈÎfi ANHP Â Ó È Ë ÙÚÈÌËÓ Î ÔÛË ÙË EÏÏËÓÈÎ AÓ ÚÔÏÔÁÈÎ EÙ ÈÚÂ. ÎÔapplefi ÂÈ ÙËÓ ÂÓËÌ ÚˆÛË ÙˆÓ È ÙÚÒÓ apple Óˆ Û ı Ì Ù appleô ÊÔÚÔ Ó ÙËÓ AÓ ÚÔÏÔÁ. T ÚıÚ appleô ËÌÔÛÈ ÔÓÙ È ÊÔ- ÚÔ Ó ÙÔÓ Â Ú ÙÔÌ ÙÔ ÂÓ È Ê ÚÔÓÙfi ÙË, applefi ÙË ÌÔÚÈ Î Î È ÁÂÓÂÙÈÎ appleïâ Ú ˆ ÙÔ ÓÂÔ Ó fiìâóô appleâ Ô ÙˆÓ appleúô ÏËÌ ÙˆÓ ÛÙÔÓ ÁËÚ ÛÎÔÓÙ Ó Ú. Ù appleâúèâ fiìâó ı appleâúèï Ì ÓÔÓÙ È Ó ÛÎÔapple ÛÂÈ, ÚıÚ Û ÓÙ ÍË, ÂÚ ÓËÙÈÎ ÂÚÁ Û Â, ÂÓ È Ê ÚÔÓÙ appleâúèûù ÙÈÎ ÏÏ Î È apple ÚÔ ÛÈ ÛÂÈ ÂÎ ÙˆÓ Ú ÛÙËÚÈÔ- Ù ÙˆÓ ÙË EÙ Èڠ̠ΠÌÂÓ Û ÌappleÔÛ ˆÓ, ÛÙÚÔÁÁ - ÏˆÓ ÙÚ appleâ ÒÓ, È Ï ÍˆÓ, appleô ı ÈÔÚÁ ÓÒÓÂÈ Ë EÙ ÈÚÂ. T ÏÔ, Ì Ûˆ ÙÔ appleâúèô ÈÎÔ ı appleúô ÏÏÔ- ÓÙ È ÍÈfiÏÔÁ ÂÚÁ Û Â ËÌÔÛÈÂ Ì Ó ÛÙ ÈÂıÓ appleâúèô ÈÎ ÂÓÒ ı Á ÓÂÙ È Î È ÂÓËÌ ÚˆÛË ÁÈ ÁÂÁÔÓfiÙ Î È ÂÎ ËÏÒÛÂÈ appleô ÊÔÚÔ Ó ÙËÓ AÓ ÚÔÏÔÁ ÛÙÔÓ EÏÏËÓÈÎfi Î È ÈÂıÓ ÒÚÔ. COPYRIGHT T ËÌÔÛÈÂ Ì Ó ÚıÚ Â Ó È È ÈÔÎÙËÛ ÙÔ appleâúèô- ÈÎÔ ANHP Î È apple ÁÔÚ ÂÙ È ÌÂÚÈÎ ÔÏÈÎ Ó ËÌÔÛ Â Û ÙÔ ˆÚ ÙËÓ ÁÁÚ ÊË Û ÁÎ Ù - ıâûë ÙÔ È ı ÓÙÔ ÓÙ ÍË. È ÙËÓ Ó apple Ú - ÁˆÁ ÂÈÎfiÓˆÓ, Û Â ˆÓ Î È appleèó ÎˆÓ apple ÈÙÂ Ù È Âapple - ÛË Û ÂÙÈÎ ÁÎÚÈÛË Î È Ó ÊÔÚ ÙË appleëá. PAMMATEIA E. KÔ ÎÎÔ, EÓ ÔÎÚÈÓÔÏÔÁÈÎfi TÌ Ì NÔÛÔÎÔÌÂ Ô E ENA BENIZE OY IAºHMI EI È Î Ù ÒÚËÛË È ÊËÌ ÛÂˆÓ ÔÈ ÂÓ È ÊÂÚfiÌÂÓÔÈ apple Ú Î ÏÔ - ÓÙ È Ó ÂappleÈÎÔÈÓˆÓÔ Ó Ì ÙËÓ EÙ ÈÚ MEDLINE, TH.: , , FAX: , e mail: medline@otenet.gr (Yapple ı ÓÔÈ: Ó ÛË M ÛÙÔÚ, XÚÈÛÙ Ó TÛ ÚÔ ).

2 2 IOIKHTIKO YMBOY IO E HNIKH AN PO O IKH ETAIPEIA POE PO : Ù Ì Ù Ó X. NÈÎÔappleÔ ÏÔ, EÓ ÔÎÚÈÓÔÏfiÁÔ ANTI POE PO : ÕÏÎË K Ú Ó Î, O ÚÔÏfiÁÔ PAMMATEA : E Ù KÔ ÎÎÔ, EÓ ÔÎÚÈÓÔÏfiÁÔ TAMIA : X ÚË AÛ ÛÙË, O ÚÔÏfiÁÔ ME H: E ÁÁÂÏ AÓ Ú Ô, EÓ ÔÎÚÈÓÔÏfiÁÔ ÂÔ ÔÛ ZÂÁÎÈÓÈ Ô, BÈÔÏfiÁÔ ÂˆÓ KÔÓÙÔÁÂÒÚÁÔ, O ÚÔÏfiÁÔ YNTAKTIKH E ITPO H IEY YNTH YNTA H :.A. A ÌfiappleÔ ÏÔ, EÓ ÔÎÚÈÓÔÏfiÁÔ ANA HPøTE :. MËÏ ÁÎÔ, M ÈÂ Ù Ú - Ó ÈÎÔÏfiÁÔ. Mapple ÚÌapple ÏÈ, O ÚÔÏfiÁÔ Y EY YNOI Y H : KÔ ÎÔ E Ù, EÓ ÔÎÚÈÓÔÏfiÁÔ M ÚÔÌÌ ÙË KˆÓ/ÓÔ, M ÈÂ Ù Ú - Ó ÈÎÔÏfiÁÔ AÚ Ó ÙË H Ë ıôïôáô Ó ÙfiÌÔ AÛÏ ÓË ÏÔ O ÚÔÏfiÁÔ B ÎË NÈÎfiÏ Ô æ ÙÚÔ BÚ ˆÓ Ô AÓ ÚÔÌ Ë EÓ ÔÎÚÈÓÔÏfiÁÔ BÏ ÛÛÔappleÔ ÏÔ B Ú Ú EÓ ÔÎÚÈÓÔÏfiÁÔ ÂˆÚÁÈ Ë ÏÔ EÓ ÔÎÚÈÓÔÏfiÁÔ È ÓÓ ÎfiappleÔ ÏÔ ÂÓÔÊÒÓ O ÚÔÏfiÁÔ K Ï Ú X ÚË BÈÔÏfiÁÔ K ÏÏÈappleÔÏ ÙË M ÈÂ Ù Ú / Ó ÈÎÔÏfiÁÔ K ÏÔ È Ô ÚË ÕÁÁÂÏÔ AÎÙÈÓÔÏfiÁÔ K Ú ÁÎÔ ÓË XÚ ÛÙÔ MÈÎÚÔ ÈÔÏfiÁÔ M Ì ÂˆÓ M ÈÂ Ù Ú / Ó ÈÎÔÏfiÁÔ Mapple Ï ÊÔ Ù XÚ ÛÙÔ M ÈÂ Ù Ú / Ó ÈÎÔÏfiÁÔ MappleÔ ÚÔ ÓË MÈ Ï O ÚÔÏfiÁÔ ÁÎ ÏÔ KˆÓÛÙ ÓÙ ÓÔ ÂÓÂÙÈÛÙ Ó Ë ËÌ ÙÚÈÔ EÓ ÔÎÚÈÓÔÏfiÁÔ apple Ì Iˆ ÓÓË EÓ ÔÎÚÈÓÔÏfiÁÔ PÔÛfiÏ ÌÔ XÚ ÛÙÔ M ÈÂ Ù Ú / Ó ÈÎÔÏfiÁÔ ÔÊÈÎ ÙË NÈÎfiÏ Ô O ÚÔÏfiÁÔ X Ù Ë Ú ÛÙÔ ËÌ ÙÚË O ÚÔÏfiÁÔ

3 3 O H IE PO Y PAºEI TÔ appleâúèô ÈÎfi ANHP, Î ÔÛË ÙË EÏÏËÓÈÎ AÓ ÔÏÔÁÈÎ EÙ ÈÚ ÂÈ ÛÙfi Ô ÙË Û Ó ÂappleÈÌfiÚʈÛË ÙˆÓ Û ÔÏÔ ÌÂÓˆÓ ÛÙÔ ÒÚÔ ÙË AÓ ÚÔÏÔÁ Î È ÙËÓ appleúô ÁˆÁ ÙÔ ÁÓˆÛÙÈÎÔ ÓÙÈÎÂÈÌ ÓÔ ÙË ÛÙÔÓ ÂÏÏËÓÈÎfi ÒÚÔ. È ÙËÓ appleú ÁÌ ÙˆÛË ÙÔ ÙÔ ÛÎÔappleÔ ËÌÔÛÈ ÔÓÙ È ÛÙÔ appleâúèô ÈÎfi: 1. AÚıÚ ÓÙ ÍË. ÓÙÔÌÂ Ó ÛÎÔapple ÛÂÈ Û Âapple Î ÈÚ Î È ÌÊÈÏÂ- ÁfiÌÂÓ ı Ì Ù, appleô ÁÚ ÊÔÓÙ È Ì appleúôùúôapple ÙË Û ÓÙ ÎÙÈÎ ÂappleÈÙÚÔapple. OÙ Ó ÂÎÊÚ Ô Ó Û ÏÏÔÁÈÎ ÙË ÓÙ ÍË ÙÔ appleâúèô ÈÎÔ, Â Ó È Ó applefiáú Ê. ÙÈ ÏÏ appleâúèappleùòûâè Â Ó È ÂÓ applefiáú Ê. 2. ÂÓÈÎ ı Ì Ù. ÂÙÈ fiìâó Ì ÙËÓ AÓ ÚÔÏÔÁ 3. AÓ ÛÎÔapple ÛÂÈ. OÏÔÎÏËÚˆÌ ÓÂ Ó Ï ÛÂÈ È ÙÚÈÎÒÓ ıâì ÙˆÓ, ÛÙÈ ÔappleÔ Â appleôáú ÌÌ ÔÓÙ È ÔÈ Û Á ÚÔÓ applefi ÂÈ. ÓÔÓÙ È ÂÎÙ Ó - ÛÎÔapple ÛÂÈ Ì ÚÈ Ô Û ÁÁÚ Ê ˆÓ. 4. EÚ ÓËÙÈÎ ÂÚÁ Û Â. KÏÈÓÈÎ ÔÎÈÌ ÌË appleâèú Ì ÙÈÎ Ú Ó appleúôôappleùèîô Ó ÚÔÌÈÎÔ Ú ÎÙ Ú, appleô appleú ÁÌ ÙÔappleÔÈ ıëî Ó Ì ÛË ÂÚ ÓËÙÈÎfi appleúˆùfiîôïïô, ÙÔ ÔappleÔ Ô Ó appleâúèáú ÊÂÙ È Ó Ï ÙÈÎ ÛÙË ÌÂıÔ ÔÏÔÁ. ÂÚÈ Ô Ó appleúˆùô ËÌÔÛÈÂ Ì Ó appleôùâï ÛÌ Ù. 5. EÓ È Ê ÚÔ Û appleâúèappleùòûâè. ÓÔÓÙ È ÂÎÙ ÚıÚ ÂÊfiÛÔÓ ÊÔ- ÚÔ Ó Ó Î È appleôï Ûapple ÓÈ ÓÔÛ Ì Ù ÓÔÛ Ì Ù ÂÌÊ Ó ÔÓÙ È È ÈÙÂ- ÚfiÙËÙ ˆ appleúô ÙËÓ ÎÏÈÓÈÎ ÙÔ ÂΠψÛË ÙËÓ ÈÂÚ ÓËÙÈÎ ÙÔ appleúôûapple Ï ÛË ÂÈ ÎÔÏÔ ıëıâ Ó ıâú appleâ ÙÈÎ ÌÂıfi  ÛË Ì ÂÏÂÁ- Ì ÓÔ ÙÔ appleôù ÏÂÛÌ. Eapple ÛË ÛÙ ÚıÚ Ù ÌappleÔÚÔ Ó Ó apple ÚÔ ÛÈ - ÛıÔ Ó appleúˆùfiù appleâ appleâúèappleùòûâè appleúô Û ÙËÛË Ì ÙÔ Ó ÁÓÒÛÙ ÙÔ appleâúèô ÈÎÔ. 6. Eapple Î ÈÚ ı Ì Ù. ÓÙÔÌË appleâúèáú Ê ÙˆÓ ÙÂÏ ٠ˆÓ applefi ÂˆÓ ÛÂ Û ÁÎÂÎÚÈÌ Ó ı Ì. 7. Ú ÎÙÈÎ applefi ÛÂÌÈÓ ÚÈ Î È ÛÙÚÔÁÁ Ï ÙÚ apple È Î ÌÂÓ applefi È Ï ÍÂÈ. 8. ÂÚ ÏË Ë ÚıÚˆÓ ÙË ÈÂıÓÔ È ÏÈÔÁÚ Ê Û ÓÔ Â fiìâóë applefi Û ÓÙÔÌÔ Û fiïèô. ËÌÔÛÈ ÔÓÙ È ÂÓ applefiáú Ê. 9. Ú ÌÌ Ù appleúô ÙË ÓÙ ÍË. ÂÚÈ Ô Ó ÎÚ ÛÂÈ ÁÈ ËÌÔÛÈÂ Ì Ó ÚıÚ, appleúfi ÚÔÌ appleôùâï ÛÌ Ù ÂÚÁ ÛÈÒÓ, apple Ú ÙËÚ ÛÂÈ ÁÈ ÓÂappleÈı ÌËÙ ÂÓ ÚÁÂÈÂ, ÎÚ ÛÂÈ ÁÈ ÙÔ appleâúèô ÈÎfi Î.Ï.apple. ËÌÔÛÈ ÔÓÙ È ÂÓ appleôáú ʈ. ÚÔËÁÔ ÌÂÓË Ù Ùfi ÚÔÓË ËÌÔÛ Â ÛË. T ÚıÚ appleô appleô ÏÏÔÓÙ È ÛÙÔ appleâúèô ÈÎfi ANHP ÂÓ ÌappleÔÚÂ Ó Ô Ó appleô ÏËı ٠Ùfi ÚÔÓ ÁÈ ËÌÔÛ Â ÛË Û ÏÏ EÏÏËÓÈÎ appleâúèô ÈÎ. TÔ ÁÂÁÔÓfi appleú appleâè Ó Â È- ÒÓÂÙ È applefi ÂappleÈÛÙÔÏ - ψÛË ÙÔ appleúòùô Û ÁÁÚ Ê appleúô ÙÔÓ È ı ÓÙ ÓÙ ÍË. Ö ÂappleÈÙÚ appleâù È Ë appleô ÔÏ ÂÚÁ ÛÈÒÓ Ì ÚÔ ÙˆÓ ÔappleÔ ˆÓ ÂÈ ËÌÔÛÈ ıâ apple ÚÔ ÛÈ Ûı Ì ÌÔÚÊ appleâú ÏË Ë Û EÏÏËÓÈÎfi ÈÂıÓ Ó ÚÈÔ. OÏ Ù ÂÈÚfiÁÚ Ê Û ÓÔ Â ÔÓÙ È applefi ÂappleÈÛÙÔÏ appleô appleôáú ÊÂÙ È applefi ÙÔÓ appleâ ı ÓÔ ÁÈ ÙËÓ ÏÏËÏÔÁÚ Ê Û ÁÁÚ Ê. H Û ÓÔ Â ÙÈÎ ÂappleÈ- ÛÙÔÏ appleú appleâè Ó appleâúèï Ì ÓÂÈ ÏˆÛË fiùè Ù ÂÈÚfiÁÚ Ê Ô Ó ÂÁÎÚÈıÂ Î È applefi fiïô ÙÔ applefiïôèappleô Û ÁÁÚ Ê ÔÈ ÔappleÔ ÔÈ Î È Û Ó appleô- ÁÚ ÊÔ Ó ÙËÓ ÂappleÈÛÙÔÏ. ÚÔÂÙÔÈÌ Û ÙÔ ÂÈÚfiÁÚ ÊÔ. H ÁψÛÛÈÎ ÔÌÔÈÔÌÔÚÊ ÙˆÓ ÚıÚˆÓ Â Ó È apple Ú ÙËÙË. T ÚıÚ appleô appleô ÏÏÔÓÙ È ÁÈ ËÌÔÛ Â - ÛË appleú appleâè Ó Â Ó È ÁÚ ÌÌ Ó ÛÙË ËÌÔÙÈÎ Î È Ì ÙÔ ÌÔÓÔÙÔÓÈÎfi Û ÛÙË- Ì. TÔ appleâúèô ÈÎfi ANHP ÂÈ appleô  ıâ ÙÔ Û ÛÙËÌ Vancouver Î È ÂÊ Ú- Ìfi ÂÈ ÙÔ ÂÏÏËÓÈÎfi appleúfiù appleô ÁÚ Ê ÈÔÈ ÙÚÈÎÒÓ ÎÂÈÌ ÓˆÓ. T ÚıÚ appleú appleâè Ó Â Ó È ÎÙ ÏÔÁÚ ÊËÌ Ó Ì ÈappleÏfi È ÛÙËÌ Û Ï Îfi ÚÙ, applefi ÙË ÌÈ appleïâ Ú ÙˆÓ ÛÂÏ ˆÓ, Ì appleâúèıòúè ÙÔ Ï È- ÛÙÔÓ 2,5 cm. T ÂÍ ÎÂÊ Ï È Ú Ô Ó ÛÂ È È ÙÂÚË ÛÂÏ : Ë ÛÂÏ Ì ÙÔÓ Ù ÙÏÔ, Ë appleâú ÏË Ë Î È ÔÈ Ï ÍÂÈ Â ÚÂÙËÚ Ô, ÙÔ Î ÌÂÓÔ, ÔÈ Â - ÚÈÛÙ Â, Ë ÁÁÏÈÎ appleâú ÏË Ë, ÔÈ È ÏÈÔÁÚ ÊÈÎ apple Ú appleôìapple, ÔÈ apple Ó - ÎÂ, ÔÈ ÂÈÎfiÓÂ Î È ÔÈ Ï ÓÙÂ ÙˆÓ ÂÈÎfiÓˆÓ. OÏ ÔÈ ÛÂÏ Â ÚÈıÌÔ - ÓÙ È, Ú ÔÓÙ applefi ÙË ÛÂÏ Ù ÙÏÔ. ÂÏ Ù ÙÏÔ. ÂÚÈÏ Ì ÓÂÈ ( ) ÙÔÓ Ù ÙÏÔ ÙÔ ÚıÚÔ, Ô ÔappleÔ Ô appleú appleâè Ó Â Ó È Û ÓÙÔÌÔ (Ì ÚÈ 12 Ï ÍÂÈ ), ( ) ÙÔ fióôì Î È ÙÔÓ Ù ÙÏÔ ÙÔ Û Á- ÁÚ Ê (-ˆÓ), (Á) ÙÔ Ú Ì ÙÔ ÂÚÁ ÛÙ ÚÈÔ, applefi ÙÔ ÔappleÔ Ô appleúô Ú ÂÙ È Ë ÂÚÁ Û Î È Ë appleúô Ï ÛË ÙÔ Û ÁÁÚ Ê, ( ) ÙÔ fióôì, ÙË È ı Ó- ÛË Î È ÙÔ ÙËÏ ÊˆÓÔ ÙÔ Û ÁÁÚ Ê ÁÈ ÏÏËÏÔÁÚ Ê Î È Ó Ù apple, (Â) appleëá appleô ÂÓ Â ÔÌ Óˆ ÂÓ Û Û Ó Î È Ô ıëû Ó ÛÙËÓ appleú ÁÌ ÙÔappleÔ ËÛË ÙË ÂÚÁ Û, (ÛÙ) Ó apple Ú Ô Ó È ÊˆÓÔ ÓÙ Ì ÙËÓ ÂÚÁ Û. ÂÚ ÏË Ë Î È Ï ÍÂÈ Â ÚÂÙËÚ Ô. H appleâú ÏË Ë ÂÓ appleú appleâè Ó appleâú - ÓÂÈ ÙÈ Ï ÍÂÈ, ÂÓÒ ÁÈ Ù Âapple Î ÈÚ ı Ì Ù Î È ÙÈ appleâúèáú Ê appleâúèappleùòûâˆó ÛıÂÓÒÓ ÙÈ Ï ÍÂÈ. È ÙÈ Ó ÛÎÔapple ÛÂÈ appleú appleâè Ó ÂÊ ÚÌfi ÔÓÙ È ÔÈ appleâúèáú ÊÈÎ appleâúèï ÂÈ (descriptive), appleô Ó Ê ÚÔ Ó Û ÓÔappleÙÈÎ fiï Ù ÎÂÊ Ï È appleô appleâúè ÂÈ ÙÔ ÚıÚÔ Î È ÛËÌ ÓÙÈÎ Û ÌappleÂÚ ÛÌ Ù. OÈ appleâúèï ÂÈ ÙˆÓ ÂÚ ÓËÙÈÎÒÓ ÂÚÁ ÛÈÒÓ appleú appleâè Ó ˆÚ ÔÓÙ È Û ٠ÛÛÂÚÈ apple Ú ÁÚ ÊÔ, ÔÈ ÔappleÔ Â Ê ÚÔ Ó Î Ù ÛÂÈÚ ÙËÓ ÎfiÏÔ ıë ÂappleÈÎÂÊ Ï. ÎÔapplefi, YÏÈÎfi M ıô Ô, AappleÔÙÂÏ ÛÌ Ù, ÌappleÂÚ ÛÌ Ù. MÂÙ ÙËÓ appleâú ÏË Ë apple Ú Ù ıâóù È 3-10 Ï ÍÂÈ ÎÏÂÈ È. OÈ Ï ÍÂÈ Ù appleú appleâè Ó ÓÙÈÛÙÔÈ Ô Ó ÛÙÔ ÈÂıÓ fiúô appleô ÚËÛÈÌÔappleÔÈ ÙÔ Index Medicus. K Â Ì Â Ó Ô. OÈ ÂÚ ÓËÙÈÎ ÂÚÁ Û Â appleôùâïô ÓÙ È Û Ó ıˆ applefi ÙËÓ EÈÛ ÁˆÁ, YÏÈÎfi Î È Ì ÁÂıÔ, AappleÔÙÂÏ ÛÌ Ù Î È ÙËÛË. H ÂÈÛ Áˆ- Á appleâúèï Ì ÓÂÈ ÙÈ apple Ú ÙËÙÂ È ÏÈÔÁÚ ÊÈÎ apple Ú appleôìapple Î È Ó - Ê ÚÂÈ ÙÔ ÏfiÁÔ ÁÈ ÙÔÓ ÔappleÔ Ô appleú ÁÌ ÙÔappleÔÈ ıëîâ Ë ÂÚÁ Û. ÙË ÌÂıÔ ÔÏÔÁ appleâúèáú ÊÂÙ È ÙÔ appleúˆùfiîôïïô, Ì ÛË ÙÔ ÔappleÔ Ô ÂÍÂ- Ï ıëîâ Ë Ú Ó. AÓ Ê ÚÔÓÙ È ÏÂappleÙÔÌÂÚÒ Ô ÙÚfiappleÔ ÂappleÈÏÔÁ ÛıÂ- ÓÒÓ ÔappleÔÈÔ appleôùâ ÏÈÎÔ, Î ıò Î È Ë Ì ıô Ô appleô ÂÊ ÚÌfiÛıËÎÂ, ÒÛÙÂ Ë È ÚÂ Ó Ó ÌappleÔÚÂ Ó Ó apple Ú ıâ applefi ÌÂÏÏÔÓÙÈÎÔ ÂÚ - ÓËÙ. ÙËÓ appleâú appleùˆûë ÂÚ ÓÒÓ appleô ÊÔÚÔ Ó ÓıÚÒappleÔ, appleú appleâè Ó ÙÔÓ ÂÙ È fiùè Ë ÚÂ Ó appleú ÁÌ ÙÔappleÔÈ ıëîâ Ì ÛË ÙËÓ YappleÔ ÚÁÈÎ applefiê ÛË AÚÈı. A6/10983/1 {ºEK 886/B } ÁÈ ÙË ÈÂÍ ÁˆÁ KÏÈÓÈÎÒÓ ÔÎÈÌÒÓ Ê ÚÌ ÎˆÓ Î È ÙËÓ appleúôûù Û ÙÔ ÓıÚÒappleÔ Î È Ë ÔappleÔ apple Ú apple ÌappleÂÈ ÛÙË È Î Ú ÍË ÙÔ EÏÛ ÓÎÈ (1975). OÈ Ê ÚÌ Î ÙÈ- Î Ô Û Â appleô ÚËÛÈÌÔappleÔÈ ıëî Ó ÛÙË ÌÂÏ ÙË appleú appleâè Ó Ó Ê ÚÔÓÙ È Ì ÙËÓ ÎÔÈÓfi ÚËÛÙË ÔÓÔÌ Û ÙÔ. ÂÚÈÁÚ ÊÂÙ È ÙÔ ÏÈÎfi appleô ÍÈÔÏÔ- Á ıëîâ Î Ù ÙË È ÚÎÂÈ ÙË ÌÂÏ ÙË Î È ÙÔ ÎÂÊ Ï ÈÔ ÔÏÔÎÏËÚÒÓÂÙ È Ì ٠ÛÙ ÙÈÛÙÈÎ ÎÚÈÙ ÚÈ appleô ÚËÛÈÌÔappleÔÈ ıëî Ó. T appleôùâï ÛÌ Ù apple ÚÔ ÛÈ ÔÓÙ È ÔÏÔÎÏËÚˆÌ Ó Î È Û ÓÙÔÌ. OÛ Ó Ê ÚÔÓÙ È Û apple Ó ÎÂ, ÂÓ Âapple Ó Ï Ì ÓÔÓÙ È ÛÙÔ Î ÌÂÓÔ. ÙË Û ÙËÛË appleâúèáú ÊÔÓÙ È ÔÈ appleúôôappleùèî appleô È ÓÔ ÁÔÓÙ È Ì ٠appleôùâï ÛÌ Ù ÙË ÌÂÏ ÙË, Î ıò Î È Ù ÙÂÏÈÎ Û ÌappleÂÚ ÛÌ Ù. ÂÓ Âapple Ó Ï Ì ÓÔÓÙ È fiû Ô Ó Ó ÊÂÚı ÛÙ appleôùâï ÛÌ Ù. Eapple ÛË, ÌappleÔÚÂ Ó Á ÓÂÈ Û ÁÎÚÈÛË Ì ٠appleôùâï ÛÌ Ù ÏÏˆÓ ÔÌÔÂÈ ÒÓ ÂÚÁ - ÛÈÒÓ. Ó ÔÓÙ È Ù appleôùâï ÛÌ Ù Ì ÙÔ ÛÙfi Ô ÙË ÌÂÏ ÙË, appleô- Ê ÁÔÓÙ È fiìˆ ı ÚÂÙ Û ÌappleÂÚ ÛÌ Ù, appleô ÂÓ appleúôî appleùô Ó applefi Ù appleôùâï ÛÌ Ù ÙË ÂÚÁ Û. E ÚÈÛÙ Â. Aapple ı ÓÔÓÙ È ÌfiÓÔ appleúô Ù ÙÔÌ, appleô Ô Ó ÔËı ÛÂÈ Ô ÛÈ ÛÙÈÎ. Ù applefiïôèapple Â Ë ÚıÚˆÓ, ÙÔ Î ÌÂÓÔ È ÌÔÚÊÒÓÂÙ È Ó ÏÔÁ Ì ÙÈ apple ÈÙ ÛÂÈ Î È ÙÔ ÛÙfi Ô ÙÔ Û ÁÁÚ Ê. ÙÈ ÂÓ È Ê ÚÔ Û appleâúèappleùòûâè ÛıÂÓÒÓ appleúôëáâ Ù È Ë ÂÈÛ ÁˆÁ Î È ÎÔÏÔ ıô Ó Ë appleâúè- ÁÚ Ê ÙË appleâúèappleùòûâˆ Î È Ë Û ÙËÛË. BÈ ÏÈÔÁÚ ÊÈÎ apple Ú appleôìapple. AÚÈıÌÔ ÓÙ È ÛÙÔ Î ÌÂÓÔ Ì ÍÔÓÙ ÚÈıÌfi, Ó ÏÔÁ Ì ÙË ÛÂÈÚ appleô ÂÌÊ Ó ÔÓÙ È.  appleâú appleùˆûë Ó ÊÔ- Ú Û ÔÓfiÌ Ù Û ÁÁÚ Ê ˆÓ ÛÙÔ Î ÌÂÓÔ, ÂÊfiÛÔÓ Â Ó È Í ÓÔÈ, ÌÂÙ ÙÔ ÂappleÒÓ ÌÔ ÙÔ appleúòùô Û ÁÁÚ Ê ÎÔÏÔ ıâ Ë Û ÓÙÔÌÔÁÚ Ê et al, ÂÓÒ ÛÙÔ EÏÏËÓÂ Û ÁÁÚ ÊÂ Î È Û Ó.. EÊfiÛÔÓ ÔÈ Û ÁÁÚ ÊÂ Â Ó È Ô, ÌÂÙ Í ÙˆÓ ÂappleˆÓ ÌˆÓ ÙÔappleÔıÂÙÂ Ù È Ë Ï ÍË Î È.

4 4 OÏ ÔÈ È ÏÈÔÁÚ ÊÈÎ apple Ú appleôìapple ÙÔ ÎÂÈÌ ÓÔ - Î È ÌfiÓÔÓ Ù - appleú appleâè Ó apple Ú Ô Ó ÛÙÔ È ÏÈÔÁÚ ÊÈÎfi Î Ù ÏÔÁÔ. O ÚÈıÌfi ÙˆÓ È ÏÈÔÁÚ ÊÈÎÒÓ apple Ú appleôìappleòó appleú appleâè Ó appleâúèôú ÂÙ È ÛÙÔÓ ÙÂÏ ˆ apple Ú ÙËÙÔ. ÙÈ Ó ÛÎÔapple ÛÂÈ, ÔÈ È ÏÈÔÁÚ ÊÈÎ apple Ú - appleôìapple ÂÓ appleú appleâè Ó Â Ó È appleâúèûûfiùâúâ applefi 100. Ù ÚıÚ ÂappleÈÎ È- ÚfiÙËÙ (Âapple Î ÈÚ ı Ì Ù, ÚıÚ ÓÙ ÍË ) ı appleú appleâè Ó Ó Ê ÚÔ- ÓÙ È ÌfiÓÔ 5-6 ÚıÚ ÌÔÓÔÁÚ Ê Â, ÁÈ Ù ÔappleÔ Ô Û ÁÁÚ Ê appleèûùâ ÂÈ fiùè Â Ó È apple Ú ÙËÙ ÁÈ ÙËÓ ÔÏÔÎÏËÚˆÌ ÓË appleïëúôêfiúëûë ÙÔ Ó ÁÓÒÛÙË ÛÙÔ ı Ì. H Û ÓÙ ÍË ÙÔ È ÏÈÔÁÚ ÊÈÎÔ Î Ù ÏfiÁÔ Á ÓÂÙ È ÚÈıÌËÙÈÎÒ, Ì ÛË ÙÔÓ ÍÔÓÙ ÚÈıÌfi Î È ÙË ÛÂÈÚ ÙˆÓ È ÏÈÔÁÚ ÊÈÎÒÓ apple Ú appleô- ÌappleÒÓ ÛÙÔ Î ÌÂÓÔ. AÓ Ê ÚÔÓÙ È Ù ÂappleÒÓ Ì Î È Ù Ú ÈÎ ÙˆÓ ÔÓÔÌ - ÙˆÓ fiïˆó ÙˆÓ Û ÁÁÚ Ê ˆÓ Ì ÚÈ ÍÈ (fiù Ó Â Ó È appleâúèûûfiùâúôè ÎÔÏÔ ıâ Ë Ó ÂÈÍË et al), Ô Ù ÙÏÔ ÙË ÂÚÁ Û, Ë Û ÓÙÔÌÔÁÚ Ê ÙÔ Ù Ù- ÏÔ ÙÔ appleâúèô ÈÎÔ, ÙÔ ÙÔ, Ô ÙfiÌÔ, Ë appleúòùë Î È Ë ÙÂÏ ٠ÛÂÏ ÙË ËÌÔÛÈ Ûˆ apple.. You CH, Lee KY, Chey WY, Menguy R. Electrogastrographic study of patients with unexplained nausea. Gastroenterology 1980, 79:  appleâú appleùˆûë appleô ÂÓ Ó Ê ÚÂÙ È fióôì Û ÁÁÚ Ê ˆ, ÛËÌÂÈÒÓÂÙ È Ë Ï ÍË AÓÒÓ ÌÔ (ÁÈ ÂÏÏËÓÈÎ ËÌÔÛ Â ÛË) Anonymous.. Anonymous. Coffe drinking and cancer of the pancreas (Editorial). Br Med J 1981, 283:628. Ú appleôìapple appleô Ó Ê ÚÔÓÙ È Û ÂÚÁ Û Â appleô ËÌÔÛÈ ÔÓÙ È ÛÂ Û ÌappleÏËÚÒÌ Ù (supplements) ÂÎ fiûâˆó, appleú appleâè Ó Û ÓÔ Â ÔÓÙ È Ì ÙÔÓ ÚÈıÌfi ÙÔ Û ÌappleÏËÚÒÌ ÙÔ, appleô ÛËÌÂÈÒÓÂÙ È Û apple Ú ÓıÂÛË, ÌÂÙ ÙÔÓ ÙfiÌÔ... Blood, 54 (Suppl 1):26.OÈ Û ÓÙÌ ÛÂÈ ÙˆÓ Ù ÙÏˆÓ ÙˆÓ appleâúèô ÈÎÒÓ appleú appleâè Ó Á ÓÔÓÙ È Ì ÛË ÙÔ Index Medicus. ÂÓ ÙÔappleÔıÂ- ÙÔ ÓÙ È ÙÂÏ  ÛÙ ÎÚÒÓ Ì ÙˆÓ Û ÁÁÚ Ê ˆÓ Î È ÛÙÈ Û ÓÙÌ ÛÂÈ ÙˆÓ appleâúèô ÈÎÒÓ. ÙË È ÏÈÔÁÚ Ê ÙˆÓ Âapple Î ÈÚˆÓ ıâì ÙˆÓ, apple Ú Ï appleô- ÓÙ È ÔÈ Ù ÙÏÔÈ ÙˆÓ ÂÚÁ ÛÈÒÓ. È ÙËÓ Î Ù ÒÚËÛË Û ÁÁÚ Ì ÙˆÓ ÌÔÓÔ- ÁÚ ÊÈÒÓ ÛÙÔ È ÏÈÔÁÚ ÊÈÎfi Î Ù ÏÔÁÔ, Ó Ê ÚÔÓÙ È ÛÙË ÛÂÈÚ Ù ÂappleÒ- Ó Ì Î È Ù Ú ÈÎ ÙˆÓ Û ÁÁÚ Ê ˆÓ, Ô Ù ÙÏÔ, Ô ÚÈıÌfi ÂÎ fiûâˆ, Ô ÂÎ fiùë, Ë applefiïë ÂÎ fiûâˆ, ÙÔ ÙÔ Î È ÔÈ ÛÂÏ Â ÙË Ó ÊÔÚ. H Ó ÊÔÚ Û ÎÂÊ Ï ÈÔ È Ï Ô appleú appleâè Ó Á ÓÂÙ È Ì ÙÔÓ ÎfiÏÔ ıô ÙÚfiappleÔ: apple ÛÈÏÂ Ô I. ÚˆÙfi ˆ. ÙÔ: ıôáfióôè Ì ÎËÙÂ Î È apple Ú ÛÈÙ. BHTA, Aı Ó, 1983: AÓ Ë È ÏÈÔÁÚ ÊÈÎ apple Ú appleôìapple appleôùâïâ ÎÂÊ Ï ÈÔ Û ÁÁÚ Ì ÙÔ appleô ÂÈ ÁÚ ÊÙ applefi ÏÏÔÓ Û ÁÁÚ Ê, Ë Ó ÊÔÚ Á ÓÂÙ È ˆ ÂÍ :Weinstein L, Swartz MN. Pathogenic properties of invading microorganisms. In: ( ÙÔ): Sodeman WA ed (;h eds ;h ÓÙ.) Pathologic Physiology. Saunders, Philadelphia, 1987: MË ËÌÔÛÈÂ Ì Ó ÂÚÁ Û Â Î ıò Î È appleúôûˆappleèî ÂappleÈÎÔÈÓˆÓ Â ÂÓ ÚËÛÈÌÔappleÔÈÔ ÓÙ È ˆ È ÏÈÔÁÚ ÊÈÎ apple Ú appleôìapple. AÚıÚ, appleô Ô Ó Á ÓÂÈ ÂÎÙ ÁÈ ËÌÔÛ Â ÛË, ÌappleÔÚÔ Ó Ó appleâúèïëêıô Ó ÛÙË È ÏÈÔÁÚ - Ê. ÙËÓ ÙÂÏ ٠appleâú appleùˆûë, ÌÂÙ ÙË Û ÓÙÔÌÔÁÚ Ê ÙÔ appleâúèô È- ÎÔ ÛËÌÂÈÒÓÂÙ È Ë Ó ÂÈÍË applefi ËÌÔÛ Â ÛË. AÁÁÏÈÎ appleâú ÏË Ë. ÂÚÈÏ Ì ÓÂÈ Ù ÔÓfiÌ Ù ÙˆÓ Û ÁÁÚ Ê ˆÓ Î È ÙËÓ È ÈfiÙËÙ ÙÔ, ÙÔÓ Ù ÙÏÔ ÙË ÂÚÁ Û Î È ÙÔ Ú Ì ÙÔ ÂÚÁ ÛÙ ÚÈÔ applefi ÙÔ ÔappleÔ Ô appleúô Ú ÂÙ È Ë ÂÚÁ Û. H appleâú ÏË Ë ÂÓ appleú appleâè Ó appleâú- ÓÂÈ ÙÈ Ï ÍÂÈ, ÂÓÒ ÁÈ Ù Âapple Î ÈÚ ı Ì Ù Î È ÙÈ appleâúèáú - Ê appleâúèappleùòûâˆó ÛıÂÓÒÓ ÙÈ Ï ÍÂÈ. È ÙÈ Ó ÛÎÔapple ÛÂÈ appleú appleâè Ó ÂÊ ÚÌfi ÔÓÙ È ÔÈ appleâúèáú ÊÈÎ appleâúèï ÂÈ (descriptive), appleô Ó Ê ÚÔ Ó Û ÓÔappleÙÈÎ fiï Ù ÎÂÊ Ï È appleô appleâúè ÂÈ ÙÔ ÚıÚÔ Î È ÛËÌ ÓÙÈÎ Û ÌappleÂÚ ÛÌ Ù. OÈ appleâúèï ÂÈ ÙˆÓ ÂÚ ÓËÙÈÎÒÓ ÂÚÁ ÛÈÒÓ appleú appleâè Ó ˆÚ ÔÓÙ È Û ٠ÛÛÂÚÈ apple Ú ÁÚ ÊÔ, ÔÈ ÔappleÔ Â Ê ÚÔ Ó Î Ù ÛÂÈÚ ÙËÓ ÎfiÏÔ ıë ÂappleÈÎÂÊ Ï. Aim, Material, Methods, Results, Coclusions. MÂÙ ÙËÓ appleâú ÏË Ë apple Ú Ù ıâóù È 3-10 Ï ÍÂÈ, apple - Ú ÙËÙ ÁÈ ÙË Û ÓÙ ÍË ÙˆÓ Â ÚÂÙËÚ ˆÓ ÙÔ appleâúèô ÈÎÔ (Key words). H appleôèfiùëù ÙˆÓ ÁÁÏÈÎÒÓ appleâúèï ÂˆÓ appleú appleâè Ó Â Ó È ÚÎÂÙ ÈÎ ÓÔappleÔÈ- ËÙÈÎ, ÂappleÂÈ appleôùâïâ ÛËÌ ÓÙÈÎfi ÎÚÈÙ ÚÈÔ appleô Ô ÙÔ appleâúèô ÈÎÔ ÛÙÔ ÈÂıÓ Π٠ÏfiÁÔ ÈÔÈ ÙÚÈÎÒÓ appleâúèô ÈÎÒÓ (Index Medicus). AÚ ıìëûë ÎÂÊ Ï ˆÓ ÛÂ Ó ÛÎÔapple ÛÂÈ, Âapple Î ÈÚ ı Ì Ù. OÏ Ù ÎÂÊ Ï È ÚÈıÌÔ ÓÙ È ÌÂ Ú ÈÎÔ ÚÈıÌÔ : 1,2,3 ÎÏapple. T appleôîâê - Ï È Ê ÚÔ Ó ÙÔÓ ÚÈıÌfi ÙÔ Ú ÈÎÔ ÎÂÊ Ï Ô, ÙÂÏÂ Î È ÎÔÏÔ ıâ Ô ÚÈıÌfi ÙÔ appleôîâê Ï Ô : 1.1., , Î.Ô.Î. Ó ÎÂ. ÎÙ ÏÔÁÚ ÊÔ ÓÙ È Ì ÈappleÏfi È ÛÙËÌ, Û ˆÚÈÛÙ ÛÂÏ. AÚÈıÌÔ ÓÙ È Ì ÙË ÛÂÈÚ appleô ÂÌÊ Ó ÔÓÙ È ÛÙÔ Î ÌÂÓÔ, ÌÂ Ú ÈÎÔ ÚÈıÌÔ. Ú appleâè Ó Ê ÚÔ Ó appleâúèâîùèî Î È Û ÓÙÔÌË ÂappleÂÍ ÁËÛË, ÒÛÙ ÁÈ ÙËÓ Î Ù ÓfiËÛ ÙÔ Ó ÌËÓ Â Ó È apple Ú ÙËÙÔ Ó Î Ù Ê ÁÂÈ Ô Ó - ÁÓÒÛÙË ÛÙÔ Î ÌÂÓÔ. K ıâ ÛÙ ÏË Ê ÚÂÈ ÂappleÂÍËÁËÌ ÙÈÎ Î È Û ÓÙÔÌË ÂappleÈ- ÎÂÊ Ï. OÈ ÂappleÂÍËÁ ÛÂÈ ÙˆÓ Û ÓÙÔÌÔÁÚ ÊÈÒÓ Î ıò Î È ÔÈ ÏÔÈapple È ÎÚÈÓ ÛÂÈ Á ÓÔÓÙ È ÛÙÔ Ù ÏÔ ÙÔ apple Ó Î. EÈÎfiÓÂ. T Û Ì Ù, Û Â È ÛÌ Ó Ì ÛÈÓÈÎ ÌÂÏ ÓË, Î È ÔÈ ÊˆÙÔÁÚ - Ê Â appleú appleâè Ó ÛÙ ÏÓÔÓÙ È ÛÙÔ appleúˆùfiù appleô, ÒÛÙÂ Ó Â Ó È Î Ù ÏÏËÏ ÁÈ ÌÂÛË ÊˆÙÔÁÚ ÊÈÎ Ó apple Ú ÁˆÁ Î È ÂÎÙ appleˆûë. ÙÔ apple Ûˆ Ì ÚÔ ÙÔ Ó ÁÚ ÊÔÓÙ È Ì ÌÔÏ È Ô ÚÈıÌfi ÙË ÂÈÎfiÓ, Ó ÏÔ appleô Ó Â ÓÂÈ ÙÔ Óˆ Ì ÚÔ Î È ÔÈ Û ÁÁÚ ÊÂ. TÔappleÔıÂÙÔ ÓÙ È ÛÂ Ê ÎÂÏÔ, Ó ÌÂÛ ÛÂ Ô ÛÎÏËÚ ÚÙfiÓÈ, ÁÈ Ó ÌËÓ ÙÛ ÎÈÛÙÔ Ó ÛÙË ÌÂÙ ÊÔ- Ú. OÈ Ù ÙÏÔÈ ÙˆÓ ÂÈÎfiÓˆÓ appleú appleâè Ó Ó ÁÚ ÊÔÓÙ È Ì ÙÔÓ ÚÈıÌfi appleô ÓÙÈÛÙÔÈ Â ÛÙËÓ ÂÈÎfiÓ Û ˆÚÈÛÙfi ÚÙ. EappleÂÍËÁ ÛÂÈ Û ÂÙÈÎ Ì ÙÈ ÂÈÎfiÓ ÌappleÔÚÔ Ó Ó Ó ÊÂÚıÔ Ó ÛÙÔÓ Ù ÙÏÔ. È ÙÔ Ì ÁÂıÔ ÙˆÓ ÂÈÎfi- ÓˆÓ Û Ì Ô Ï ıâ Ù ÙÔ Û Ì ÙÔ appleâúèô ÈÎÔ. EÊfiÛÔÓ ÚËÛÈÌÔappleÔÈÔ - ÓÙ È ÊˆÙÔÁÚ Ê Â ÛıÂÓÒÓ, ÙÔ appleúfiûˆappleô ÂÓ appleú appleâè Ó Ê ÓÂÙ È. ÙËÓ ÓÙ ıâùë appleâú appleùˆûë ÂappleÈ ÏÏÂÙ È ÁÁÚ ÊË Û ÁÎ Ù ıâûë ÙÔ ÛıÂÓÔ ÁÈ ÙË ËÌÔÛ Â ÛË ÙË ÊˆÙÔÁÚ Ê. OÏ ÔÈ ÂÈÎfiÓÂ Ó Ê ÚÔÓÙ È ÛÙÔ Î ÌÂÓÔ Î È ÚÈıÌÔ ÓÙ È ÌÂ Ú ÈÎÔ ÚÈıÌÔ. OÓÔÌ ÙÔÏÔÁ. OÈ Û ÁÁÚ Ê appleú appleâè Ó ÚËÛÈÌÔappleÔÈÔ Ó ÙÔ apple ÁÎÔ- ÛÌ ˆ apple Ú ÂÎÙÔ Ù ÙÏÔ. È ÙËÓ ÂappleÈÏÔÁ ÙˆÓ fiúˆó Î È ÙˆÓ ÔÓÔÌ - ÙˆÓ (Ô ÛÈÒÓ, ÔÓÙÔÙ ÙˆÓ, ÔÚÁ ÓÈÛÌÒÓ, ÓÔÛËÌ ÙˆÓ Î.Ï.apple.), ÎÚ ÓÂÙ È ÛÎfiappleÈÌÔ ÔÈ Û ÁÁÚ ÊÂ Ó Û Ì Ô Ï ÔÓÙ È ÙÔ ÂÍÈÏfiÁÈÔ BÈÔÈ ÙÚÈÎ OÚÔÏÔÁ, MeSH-E A. EÎ ÔÛË IATPOTEK, Aı Ó, MÂÙÚ ÛÂÈ. MÂÙÚ ÛÂÈ Ì ÎÔ, Ô, ÚÔ Î È fiáîô appleú appleâè Ó Ó Ê ÚÔÓÙ È Û ÌÂÙÚÈÎ ÌÔÓ Â (Ì ÙÚÔ, ÈÏ., Ï ÙÚÔ) ÛÙÈ appleô È È- Ú ÛÂÈ ÙÔ. OÈ ıâúìôîú Û Â appleú appleâè Ó ÓÔÓÙ È Û ıìô KÂÏÛ Ô. OÈ ÚÙËÚÈ Î appleè ÛÂÈ appleú appleâè Ó ÓÔÓÙ È Û ÈÏÈÔÛÙ ÛÙ ÏË Ú ÚÁ - ÚÔ. ÈfiÚıˆÛË Ù appleôáú ÊÈÎÒÓ ÔÎÈÌ ˆÓ. Ú ÁÌ ÙÔappleÔÈÂ Ù È Ì ÊÔÚ applefi ÙÔ Û ÁÁÚ ÊÂ. EÎÙÂÙ Ì Ó ÌÂÙ ÔÏ ÂÓ Á ÓÔÓÙ È ÂÎÙ. AÓ Ù apple. Aapple ÁÔÚ ÂÙ È Ë ÊˆÙÔÙ appleèî Ó apple Ú ÁˆÁ ÙˆÓ ËÌÔÛÈÂ Ì - ÓˆÓ ÂÚÁ ÛÈÒÓ. H appleúôì ıâè applefi ÙÔ Û ÁÁÚ ÊÂ Ó Ù appleˆó Á ÓÂÙ È appleôîïâèûùèî applefi ÙËÓ ÂÙ ÈÚ MEDLINE. OÈ Û ÁÁÚ Ê ÂappleÈ Ú ÓÔ- ÓÙ È Ì ÙÔ ÎfiÛÙÔ ÙÔ. T Ó Ù apple apple Ú ÁÁ ÏÏÔÓÙ È Î Ù ÙË ÈfiÚıˆ- ÛË ÙˆÓ ÔÎÈÌ ˆÓ. XÂÈÚfiÁÚ Ê ÂÚÁ ÛÈÒÓ appleô ËÌÔÛÈ ÔÓÙ È, ÂÓ ÂappleÈÛÙÚ ÊÔÓÙ È ÛÙÔ Û ÁÁÚ ÊÂ. YappleÔ ÔÏ ÂÈÚÔÁÚ ÊÔ :T ÂÈÚfiÁÚ Ê appleôûù ÏÏÔÓÙ È ÛÙË È ı ÓÛË:.A. A AMO OY O EPIO IKO ANHP EN OKPINO O IKO TMHMA- N MAIEYTHPIO E ENA BENIZE OY Ï. E. BENIZE OY A HNA H ÂÚÁ Û Ù ÚÔÌÂ Ù È ÛÂ Ê ÎÂÏÔ applefi ÔÓÙÚfi ÚÙ, ÂÛˆÎÏ ÔÓÙ ÙÈ ÊˆÙÔÁÚ Ê Â Î È ÙË ÈÛÎ ÙÙ (ÂÊ fiûôó apple Ú ÂÈ) Ì Û Û ÛÎÏËÚfi ÚÙfiÓÈ. E Ó Ë appleôûùôï Á ÓÂÙ È Ì Ûˆ ÙˆÓ EÏÏËÓÈÎÒÓ T ÚÔÌ ˆÓ Ó ÌËÓ ÎÔÏÔ ıâ Ù È Û ÛÙËÌ ÓË È ÈÎ Û.

5 5 E P I E X O M E N A 6 ËÌ ˆÌ Û ÓÙ ÍË 7 N Ô Ì Ô ÏÈÔ - N Ô ÂÎ ÓËÌ 9 AÊÈ ÚˆÌ : Y O THPIZOMENH ANA APA ø H THN AN PIKH Y O ONIMOTHTA (M ÚÔ 2Æ) Treatment of Non-Obstructive Azoospermia with Ooplasmic Injections of Immature Germ Cells Recent Advances and update in Assisted Reproductive Technologies Multiply Pregnancies after Assisted Reproductive Technologies (ART) Transplantation of Human Germ Cells into Immunosuppressed Animal Testicles for the Management of Non-Obstructive Azoospermia The Impact of Cigarette Smoking on Male Fecundity: Its Effects on Sperm Characteristics and Sexual Behavior 37 AÓ ÛÙÚ ÈÌË YappleÔÁÔÓÈÌfiÙËÙ, º ÚÌ Î ÙÈÎ Î È A ÙfiÌ ÙË, Û ÕÓ Ú Ì Ÿ ÈÌË ŒÓ ÚÍË ÁÁÂÓ YappleÂÚappleÏ Û ÙˆÓ EappleÈÓÂÊÚÈ ˆÓ, ÏfiÁˆ AÓÂapple ÚÎÂÈ ÙË 21- ÚÔÍ Ï ÛË 42 Report on Andrology Course in Athens 2Æ TEYXO, 3 o TOMO AÓ ÛÎÔapple ÛÂÈ ñaïïëïâapple Ú ÛË ÂÓ ÔÎÚÈÓÈÎÔ Î È ÓÔÛÔÏÔÁÈÎÔ Û ÛÙ Ì ÙÔ ñh ÔÛÙÂÔapplefiÚˆÛË ÛÙÔÓ Ó Ú EÚ ÓËÙÈÎ ÂÚÁ Û Â EÓ È Ê ÚÔÓÙ appleâúèûù ÙÈÎ

6 6 HMEIøMA TH YNTA H M ÚÙÈÔ 2001 M ÙÔ apple ÚfiÓ ÙÂ Ô ÔÏÔÎÏËÚÒÓÂÙ È ÙÔ ÊÈ ÚˆÌ ÛÙËÓ appleôûùëúè fiìâóë Ó apple Ú ÁˆÁ ÁÈ ÙËÓ Ó ÚÈÎ appleôáôóèìfiùëù. Aapplefi ÙËÓ appleô Ô ÙÔ 1 Ô Ì ÚÔ, ÔÌ ÙË Â ÈfiÙËÙ fiùè ı apple ÚÍÂÈ Ó ÏÔÁË ÂappleÈÙ ÛÙÔ appleôïâèapplefiìâóô Ì ÚÔ ÙÔ ÊÈÂÚÒÌ ÙÔ, appleô ÁÈÓ Ì ÙËÓ appleúôûapple ıâè ÙÔ ÂÍ ÚÂÙÔ Î È È appleúâappleô Û Ó ÏÊÔ, Î ıëáëùô N. ÔÊÈÎ ÙË. È fiûô appleúôû ÂÙ ٠ÌÈÎÚ ÁÚ ÌÌ Ù ÙË Î ÔÛË ı apple Ú ÙËÚ Û Ù fiùè apple Ú Ô Ó Î appleôèâ ÏÏ Á ÛÙËÓ ÂÎ ÔÙÈÎ ÔÌ. Ú ÁÌ ÙÈ, ÌÂÙ ÙËÓ Ó Ó ˆÛË ÙË ÂÌappleÈÛÙÔÛ ÓË ÙÔ Ó Ô.. ÙË EÙ ÈÚÂ, ÎÚ ıëîâ Ó ÁÎ Ë ÌÂÚÈÎ ÏÏ Á ÛÙË Û ÓÙ ÎÙÈÎ ÂappleÈÙÚÔapple ÙÔ appleâúèô ÈÎÔ Î È ÙÔ appleâ ı ÓÔ ÏË. ŒÙÛÈ appleâ ÒÚËÛ Ó ÂÎÏÂÎÙÔ Û Ó ÂÏÊÔÈ Ì ÈÛÙÔÚÈÎ appleúôûêôú ÛÙËÓ EÙ ÈÚÂ Î È ÙÔ appleâúèô ÈÎfi, ÙÔ ÔappleÔ Ô Â ÚÈÛÙÔ Ì ıè ÁÈ ÙË Ô ıâè ÙÔ. ˆÚÔ Ì ÙËÓ appleô Û ÙÔ ÌfiÓÔ appleúôûˆúèó Î È ÙË Ó Û ÛË ÙÔ Ì ÙÔ appleâúèô ÈÎfi, Î Ù ÛÙ ÛË Ó ÌÔÓ. E Ì ÛÙ appleôï Ï ÁÔÈ Î È ÚÂÈ fiì ÛÙ fiïôè. M ÙËÓ Â Î ÈÚ Î ÏÔÛˆÚ Ô ÌÂ Ù Ó Ì ÏË Î È Â fiì ÛÙÂ Ë appleúôûêôú ÙÔ Ó ÌappleÔÚ ÛÂÈ Ó Êı ÛÂÈ ÂΠÓË ÙˆÓ appleâú ÔÌ ÓˆÓ ÌÂÏÒÓ. Aapplefi Ì ÚÔ Ì, ı apple ÚÍÂÈ appleúôûapple ıâè Ó ÍÈÔappleÔÈËıÔ Ó fiïôè ÔÈ Û Ó ÂÏÊÔÈ ÛÙÔ Ì ÙÚÔ appleô ÙÔ ÂappleÈÙÚ appleô Ó ÔÈ Û Óı ÎÂ. M ÙËÓ Â Î ÈÚ ı ıâï Ó apple Ú Î Ï Ûˆ Ù Ì ÏË ÙË Û ÓÙ ÎÙÈÎ ÔÌ Ó ÔËı ÛÔ Ó ÛÙËÓ Ó appleù ÍË ÙˆÓ Ú ÛÙËÚÈÔÙ ÙˆÓ ÙÔ appleâúèô ÈÎÔ Î Ù ı ÓÔÓÙ Ì ÚÔ ÙË Û ÁÁÚ ÊÈÎ ÙÔ appleúôûapple ıâè ÛÙÔ ANHP, ÏÏ Î È Î ÏÒÓÙ ÔÌ Â Î È Û Ó ÏÊÔ Ì ÂÚ ÓËÙÈÎfi ÎÏÈÓÈÎfi ÂÓ È Ê ÚÔÓ ÛÙÔ ÒÚÔ Ó appleâ ı ÓıÔ Ó ÛÙÔ appleâúèô ÈÎfi appleúôapple Á Ó ÔÓÙ ÙÔ ÛÎÔappleÔ ÙÔ. ÙË ı ÛË ÙˆÓ ÂappleÈÌÂÏËÙÒÓ ÏË ı È - appleèûùòûâùâ ÙËÓ apple ÚÔ Û Ó Ô Ì ÙÔ appleô ÂÎappleÚÔÛˆappleÂ Ù È applefi ÙËÓ Î. E Ù KÔ ÎÎÔ, ÂÓ ÔÎÚÈ- ÓÔÏfiÁÔ, Î È applefi ÙÔÓ Î. KˆÓÛÙ ÓÙ ÓÔ M ÚÔÌÌ ÙË, Á Ó ÈÎÔÏfiÁÔ. K È ÔÈ Ô È ı ÙÔ Ó ÁÓÒÛË Î È ÂÓıÔ ÛÈ ÛÌfi, appleô Â Ó È apple Ú ÙËÙ ÁÈ ÙËÓ ÂappleÈÙ ÙË appleúôûapple ıâè. T ÏÔ Â ÚÈÛÙÒ ÙËÓ appleâú fiìâóë ÂappleÈÌÂÏ ÙÚÈ ÏË Î.. NÈÎÔappleÔ ÏÔ ÁÈ ÙË Û ÓÔÏÈÎ appleúô- ÛÊÔÚ ÙË Î Ù ÙË ÛÎÔÏË appleâú Ô Ô ÙË Ú ÙË Î ÔÛË ÙÔ appleâúèô ÈÎÔ. H Û ÓÂ Ô ıâè ÙË applefi ÙË ı ÛË ÙË ÚÔ ÚÔ ÙË EÙ ÈÚ ı apple Ú Ì ÓÂÈ Ô ÛÈÒ Ë. TÔ ÂapplefiÌÂÓÔ ÙÂ Ô ÙÔ appleâúèô ÈÎÔ ı appleôùâï ÛÂÈ Ó È ÏÂÈÌÌ ÛÙÔÓ ÂappleÈÙ ıâûìfi ÙˆÓ appleúôûîâîïëì ÓˆÓ ÂÎ ÔÙÒÓ. AÌ Ûˆ ÌÂÙ ı Âapple Ó ÏıÔ Ì ÛÙË Û ÓËıÈÛÌ ÓË Ù ÎÙÈÎ ÙˆÓ ıâì ÙÈÎÒÓ ÂappleÈÏÔÁÒÓ Ì appleâ ı ÓÔ Î È Î Ù ÍÈˆÌ ÓÔ ÛÙÔ ÓÙÈΠÌÂÓfi ÙÔ Û Ó ÏÊÔ..A. A ÌfiappleÔ ÏÔ

7 AÓ Ú, (3), NEO YMBOY IO NEO EKINHMA AÁ appleëùô Û Ó ÂÏÊÔÈ, OÈ ÂÎÏÔÁ ÙË 10Ë M ÚÙ Ô Ó ÂÈÍ Ó ÙÔ Ó Ô ÈÔÈÎËÙÈÎfi Û Ì Ô ÏÈÔ, ÙÔ 8Æ Î Ù ÛÂÈÚ Ó applefi ÙËÓ Ú ÛË ÙË EÏÏËÓÈÎ AÓ ÚÔÏÔÁÈÎ EÙ ÈÚÂ, Ë ÔappleÔ Ê ÙÔ Û ÌappleÏ ÚˆÛ 14 ÚfiÓÈ apple ÚÔ Û ÙË ÛÙËÓ ÂÏÏËÓÈÎ È ÙÚÈÎ ÎÔÈÓfiÙËÙ. MappleÔÚ ΠÓÂ Ó appleâè, fiùè Ë EÙ ÈÚÂ È Ú ÂÙ È ÙËÓ ÂÊË Â ÙË, ÌÈ appleâú Ô Ô È È ÙÂÚ Â - ÛıËÙË Î È Î ıôúèûùèî ÛÙËÓ appleèô apple Ú ÂÍ ÏÈÍ ÙË. E Ó È ÎÔÈÓ Ë ÛıËÛË fiùè apple Ú ÙÈ ÂÁÁÂÓ ÛÎÔÏ Â Î È ÙËÓ appleâúèôúèûì ÓË ÔÈÎÔÓÔÌÈÎ ÙË Â ÚÂÈ, Ë EÙ ÈÚ Π٠ÊÂÚ fi È ÌfiÓÔ Ó ÓÂÈ Ó Û ÓÂ Î È ÍÈfiÏÔÁÔ ÂappleÈÙË- ÌÔÓÈÎfi apple ÚfiÓ, ÏÏ Î È Ó ÈÔÚÁ ÓÒÛÂÈ Ù ÛÛÂÚ ÂappleÈÙ apple ÓÂÏÏ ÓÈ Û Ó ÚÈ Î È ÙÔ ÛËÌ ÓÙÈÎfiÙÂÚÔ Ûˆ, Ó ÂÎ ÒÛÂÈ Ó appleâúèô ÈÎfi, ÙÔ ANHP, appleô appleôùâïâ ÌÈ Û Ó ÚÔ ÂÓËÌ ÚˆÛË ÛÙÔ ÒÚÔ ÙË AÓ ÚÔÏÔÁ. B ÛÈÎ È ÈÔÌÔÚÊ ÙË EÙ ÈÚÂ Ì Â Ó È Ë ÂÙÂÚÔÁ ÓÂÈ ÙˆÓ ÂÈ ÈÎÔÙ ÙˆÓ appleô ÙËÓ appleôùâïô Ó, ÂÙÂÚÔÁ - ÓÂÈ Ó ÏÔÁË Ù ÙÔ ÓÙÈÎÂÈÌ ÓÔ ÙË. E Ó È Û Ó ËÛË Û fiïô ÙÔ Û ÔÏÔ ÌÂÓÔ ÛÙÔ ÒÚÔ ÙË, fiùè Ô È ÏÔÁÔ ÌÂÙ Í ÙˆÓ È ÊfiÚˆÓ ÂÈ ÈÎÔÙ ÙˆÓ appleô Û ÔÏÔ ÓÙ È Ì ÙÔÓ Ó Ú Â Ó È apple Ú ÙËÙÔ, ÁÈ Ù Î Ì ÂÈ È- ÎfiÙËÙ, ÙÔ Ï ÈÛÙÔÓ Ì ÙË ÌÔÚÊ ÙË ÛËÌÂÚÈÓ, ÂÓ Î Ï appleùâè ÙÔ ÒÚÔ. ÕÏψÛÙÂ Ó applefi ÙÔ ÛÈÎfiÙÂÚÔ ÏfiÁÔ Ú ÛË ÙË EÙ ÈÚÂ, ÙfiÛÔ ÛÙÔÓ EÏÏËÓÈÎfi ÒÚÔ, fiûô Î È ÈÂıÓÒ, Ù Ó Ë Ó ÁÎË Û ÓÂ Ô ÂÎapple  - ÛË, È ÏfiÁÔ Î È ÂÓËÌ ÚˆÛË fiïˆó Ì. T apple Ú apple Óˆ Â Ó È ÁÓˆÛÙ Î È appleôù ÏÂÛ Ó ÙÔÓ Î ÚÈÔ ÍÔÓ appleúôáú ÌÌ ÙÈÛÌÔ fiïˆó ÙˆÓ Ú ÛÙËÚÈÔÙ - ÙˆÓ ÙË EÙ ÈÚÂ Ì ÚÈ Û ÌÂÚ. TÔ Ó Ô ÈÔÈÎËÙÈÎfi Û Ì Ô ÏÈÔ ı Û Ó ÛÂÈ ÙËÓ appleúôûapple ıâè. ŸÌˆ, ÂÎÌÂÙ Ï- Ï fiìâóô ÙËÓ Ì ÚÈ Û ÌÂÚ appleôîùëıâ Û ÂÌappleÂÈÚ, ı appleúôûapple ı ÛÂÈ Ó ÈÔÚıÒÛÂÈ Î È Û ÌappleÏËÚÒÛÂÈ Î appleôèâ appleú ÎÙÈÎ Ù ÎÙÈÎ. ŒÙÛÈ appleôê ÛÈÛÂ Ó È ÎÚ ÓÂÈ ÙÈ ÂÎapple È Â ÙÈÎ Ú ÛÙËÚÈfiÙËÙ ÛÂ Ô Î ÎÏÔ. O appleúòùô Î ÎÏÔ, ı appleâ ı ÓÂÙ È Û fiïô ÙÔ È ÙÚÈÎfi ÎÔÈÓfi Ì ÙÔ Û ÂÙÈÎfi ÂÓ È Ê ÚÔÓ ÛÙÔ ÒÚÔ, apple ÚÔ - ÛÈ ÔÓÙ ÓÂÒÙÂÚ ÂÍÂÏ ÍÂÈ ı Ì Ù ÂÈ ÈÎfiÙÂÚÔ ÂÓ È Ê ÚÔÓÙÔ. ÙË Â ÙÂÚË appleâú appleùˆûë ı Û ËÙ ÛÂÈ Î È Û ÓÂÚÁ Û Ì ÙËÓ È ÙÚÈÎ ÂÙ ÈÚ ÙÔ ÓÙ ÛÙÔÈ Ô ÒÚÔ. O Î ÎÏÔ Ùfi ÔÓÔÌ ÛÙËΠӠÂÎapple  ÛË ÛÙËÓ AÓ ÚÔÏÔÁ Î È ı appleôùâïâ ÙË Û Ó ÂÈ ÙˆÓ Ì ÚÈ Û ÌÂÚ Ú ÛÙËÚÈÔÙ ÙˆÓ ÙË. O  ÙÂÚÔ Î ÎÏÔ, ı appleâ ı ÓÂÙ È Î Ù Î ÚÈÔ ÏfiÁÔ ÛÙÔ ÓÂfiÙÂÚÔ Û Ó ÏÊÔ Ì ÂÓ È Ê ÚÔÓ ÛÙÔ ÒÚÔ, Û ÓÙÔÓ ÔÓÙ Ì ÛÊ ÈÚÈÎ ÂÎapple Â Û ÙÔ applefi fiïâ ÙÈ ÂÌappleÏÂÎfiÌÂÓ ÂÈ ÈÎfiÙËÙÂ

8 8 (EÓ ÔÎÚÈÓÔÏÔÁ, O ÚÔÏÔÁ, AÓ apple Ú ÁˆÁ, EÚÁ ÛÙ ÚÈÔ ÎÏapple.). appleâúèï Ì ÓÂÈ ıâˆúëùèîfi ÏÏ Î È appleú ÎÙÈÎfi Ì ÚÔ Î È ı Î Ï ÂÈ fiï Ù ı Ì Ù ÙË ÎÏÈÓÈÎ AÓ ÚÔÏÔÁ. O Î ÎÏÔ Ùfi appleô ÔÓÔÌ ÛÙËΠÛÙËÌ ÙÈÎ EÎapple  ÛË ÛÙËÓ KÏÈÓÈÎ AÓ ÚÔÏÔÁ appleôùâïâ ÙËÓ appleúòùë Û ÂÙÈÎ appleúôûapple ıâè, Û ÂÙ È Â ÛÙËÓ appleôîùëıâ Û ıâùèî ÂÌappleÂÈÚ applefi ÙËÓ Ó ÏÔÁË ÂÎapple  ÛË ÛÙËÓ AÓ ÚÔÏÔÁ ÛÙÔ Ô applefiùúôêô ÙË EÙ ÈÚ applefi ÒÚ ÙË AÊÚÈÎ. M ÏÈÛÙ ÛÙÔ ÙÂ Ô Ùfi ÊÈÏÔÍÂÓÂ Ù È Î È Ë Â ÚÈÛÙ ÚÈ ÂappleÈÛÙÔÏ ÙÔ ÙÂÏ ٠Ô, È ÙÚÔ Î. Kapona applefi ÙËÓ T Ó Ó, appleô ÂÎapple È Â ÙËΠapplefi ÙËÓ EÙ ÈÚ Û Aı Ó Î È ÂÛÛ ÏÔÓ ÎË, ÛÙÔ È - ÛÙËÌ 10 Ô -12 Ô /00. O Î ÎÏÔ Ùfi appleô ÊÈÏÔ ÔÍÂ Ó Á ÓÂÈ ıâûìfi, ı ÒÛÂÈ ÙËÓ Â Î ÈÚ Û appleôïïô Ó Ô Û Ó ÏÊÔ Ó appleôîù ÛÔ Ó ÌÈ ÌÈÎÚ ÂÌappleÂÈÚ ÛÙÔ ÒÚÔ ÙË AÓ ÚÔÏÔÁ, Ó Û ÛÙËÌ ÙÔappleÔÈ ÛÔ Ó ÙÈ ÓÙ - ÛÙÔÈ Â ÁÓÒÛÂÈ ÙÔ Î È Ûˆ, Î Ùˆ applefi appleúô appleôı ÛÂÈ appleô ı ÂÍÂÙ ÛıÔ Ó, Ó apple ÚÔ ÛÈ ÛıÔ Ó, fiûôè ıâï - ÛÔ Ó, ÛÙÈ ÂÍÂÙ ÛÂÈ ÙË E Úˆapple Î AÎ ËÌ AÓ ÚÔÏÔÁ. TÔ AÓ Ï ÙÈÎfi appleúfiáú ÌÌ ÙˆÓ ÂÎ ËÏÒÛÂˆÓ ÙˆÓ Ô Î ÎÏˆÓ ı ÁÓˆÛÙÔappleÔÈËıÂ Û ÓÙÔÌ Î È Ì Û applefi ÙË ÛÂÏ ÙË EÙ ÈÚ ÛÙÔ appleâúèô ÈÎfi, appleô applefi ÙÒÚ ÂÁÎ ÈÓÈ ÂÙ È. H ÛÂÏ Ù ı ÓÂÈ ÙËÓ Â Î ÈÚ Ù ÎÙÈÎ ÂappleÈÎÔÈÓˆÓ ÙÔ ÈÔÈÎËÙÈÎÔ Û Ì Ô Ï Ô, fi È ÌfiÓÔ ÌÂ Ù Ì ÏË ÙË EÙ ÈÚÂ, ÏÏ Î È ÙÔ applefiïôèappleô Ó - ÁÓÒÛÙÂ. ºÈÏÔ ÔÍ ÙÔ Âapple ÛË Â Ó È Ë ËÌÈÔ ÚÁ ÈÛÙÔÛÂÏ ÛÙÔ È ÎÙ Ô ÂÁÎ ÈÓÈ ÔÓÙ ÙÔÓ ÙÚfiappleÔ ÙË Û Á ÚÔÓË ÂappleÈÎÔÈÓˆÓ. T ÏÔ, ÁÈ fiûô ÂÓ È Ê ÚÔÓÙ È Ó ÂÓÈÛ ÛÔ Ó ÂÓÂÚÁËÙÈÎ ÙÈ Ú ÛÙËÚÈfiÙËÙ ÙË EÙ ÈÚÂ Î È ı ÏÔ Ó Ó ÁÚ ÊÔ Ó ˆ Ì ÏË ÙË, Ó Ù ÎÙ È ÛÙ Ì Ù ı ÛÙ ÏÓÂÙ È Ì Ì ÙÔ appleâúèô ÈÎfi, ÓıÂÙÔ ÙËÛË ÂÁÁÚ Ê Ì ÏÔ. Aapple Ú ÙËÙË appleúô applefiıâûë ÁÈ ÙËÓ ÂÁÁÚ Ê, Ë Û Ó appleôûùôï Û ÓÙÔÌÔ ÈÔÁÚ ÊÈÎÔ ÛËÌÂÈÒÌ ÙÔ. H ÂappleÈ Ú ÓÛË ÙË ÁÔÓÈÌfiÙËÙ ÛÙ ÓÂappleÙ ÁÌ Ó ÎÚ ÙË, apple Ú ÏÏËÏ Ì ÙÔÓ appleâúappleïëı ÛÌfi Î È ÙËÓ Ó - ÁÎ ÈfiÙËÙ ÂÏ Á Ô ÙÔ ÛÙ Ó appleù ÛÛfiÌÂÓ ÎÚ ÙË ÏÏ Î È Ë È apple ÛÙˆÛË fiùè Ë ÍËÛË ÙÔ ÔÚ Ô ÂappleÈ ˆÛË ËÌÈÔ ÚÁÂ Ó Ó ÔÏÔ Ó Î È appleâúèûûfiùâúô ÁËÚ ÛÎÔÓÙ appleïëı ÛÌfi ÛÙÔÓ appleï Ó ÙË Ì, fiï Ù Û Ó ÛÌ Ó Î È Ì ÙË È ÚÔÓÈÎ ÓËÛ ÙÔ ÚÔÌËı, Ô ËÁÔ Ó ÛÙË Û Ó ÈÂ Ú ÓÛË ÙˆÓ ÁÓÒÛÂÒÓ Ì ÛÙ È ÊÔÚ ı Ì Ù. TÔ Ó ÙÔ ÚÓÈ Ù È Ùfi Î Ó ÂÓ ÙÔ ÏÏ ÂÈ Î È Ô ÓfiÌÔ ÙË Ó ÁÎ ÈfiÙËÙ Â Ó È ÛÒappleËÙÔ. H AÓ ÚÔÏÔÁ applefi Ó ÌÈÎÚfi ÒÚÔ ÚÔÌ ÓÙÈÎÒÓ ÂÚ ÓËÙÒÓ, Á ÓÂÙ È ÁÓÒÛË ÙË Î ıëìâúèó È ÙÚÈÎ appleú ÍË. K È Ô ÒÚÔ ÌÂÁ ÏÒÓÂÈ. H EÏÏËÓÈÎ AÓ ÚÔÏÔÁÈÎ EÙ ÈÚ ÂappleÈı ÌÂ Ó ÌÂÁ ÏÒÛÂÈ ÂÊÔ È ÛÌ ÓË Ì ÁÓÒÛÂÈ Î È Â ÈÛıËÙÔappleÔ ËÛË. Ùfi ËÙ ÙË ÈÎ Û Û Ó ÚÔÌ Î È Û ÓÂÚÁ Û. OÈ ÂÎ ËÏÒÛÂÈ ÙË Â Ó È ÌÈ Â Î ÈÚ ÁÈ fiïô! È ÙÔ ÈÔÈÎËÙÈÎfi Ì Ô ÏÈÔ H appleúfiâ ÚÔ.X. NÈÎÔappleÔ ÏÔ

9 AÓ Ú, (3), AºIEPøMA MEPO 2Æ TREATMENT OF NON-OBSTRUCTIVE AZOOSPERMIA WITH OOPLASMIC INJECTIONS OF IMMATURE GERM CELLS NIKOLAOS SOFIKITIS ASSISTANT PROFESSOR, DEPARTMENT OF UROLOGY, TOTTORI UNIVERSITY SCHOOL OF MEDICINE, YONAGO, JAPAN SPERMIOGENESIS Spermiogenesis is the sequence of cytological events that result in the formation of mature spermatozoa from round spermatids. In most of the mammals, this process takes place within the seminiferous tubule throughout the reproductive life span of the male. During spermiogenesis process no cell division is involved. This process is a metamorphosis in which a round cell is converted into a highly motile structure. The major changes can be grouped into the formation of acrosome, nuclear changes, development of the flagellum, and reorganization of the cytoplasm. The acrosome structure arises from the Golgi complex. Proacrosomal granules are established in the Golgi complex. They coalesce to form a single, large granule that comes into contact with the nuclear membrane and spreads over 25 to 60% of the nuclear surface. The male gamete DNA becomes stabilized during spermiogenesis and becomes resistant to digestion by the enzyme DNAase. This stabilization occurs at a time when lysine-rich histones are being replaced by arginine-rich testis specific histones in the spermatid nuclei. At the stage of the elongated spermatid the histones of round spermatids are replaced by protamines. The transition proteins determined in elongated spermatids consist of two major types (Fawcett et al., 1971). They occupy the nucleus between the removal of histones and their replacement by protamines. After fertilization the sperm DNA becomes devoid of protamines again and associated with histones of oocyte origin (Perreault and Zirkin, 1982). The central core of the sperm tail, the axial filament, develops from the pair of centrioles located at the periphery of the spermatid cytoplasm and subsequently moves centrally to be lodged at the caudal pole of the nucleus. The basic structure of the axial filament is common to flagella and cilia and consists of nine peripheral double microtubules. The last steps in spermiogenesis are characterized by additional changes in the relationship of the nucleus and cytoplasm and movement of organelles within the spermatids. EVOLUTION OF OOPLASMIC INJECTIONS OF ROUND SPERMATIDS IN ANIMALS Considering that the round spermatid contains a haploid set of chromosomes and a haploid amount of DNA several investigators evaluated the reproductive potential of the round spermatid. Ogura and Yanagimachi (1993) have shown that round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. In another studies Ogura et al. (1993 and 1994) found that round spermatid nuclei incorpotated into mature oocytes via electrofusion commonly failed to develop into large pronuclei. However, they showed that when mouse intact round spermatids are

10 10 Nikolaos Sofikitis successfully fused with oocytes some of the resulting zygotes develop into normal offspring (Ogura et al.,1994). To avoid oocyte damage due to the fusion process we chose a microsurgical approach to transfer round spermatid nuclei into rabbit ooplasm (Sofikitis et al ). By this method we achieved three pregnancies. In another study we showed that electrical stimulation of oocytes prior to ooplasmic injections of rou nd spermatids has beneficial effects on oocyte activation, fertilization, and embryonic and fetal development and results in 13% live birth rate per activated oocyte (Sofikitis et al., 1996a). We also showed that testicular round spermatids recovered from animals with primary testicular dam age (i.e., varicocelized rabbits) have imp aired reproductive potential (Sofikitis et al., 1996b). The latter study raised the probability that animals or humans with primary testicular failure m ay not have anatomically and physiologically normal round spermatids. In contrast, Sasagawa and Yanagimachi (1997) achieved delivery of healthy offspring after ooplasmic injections of round spermatid nuclei recovered from cryptorchid mice. Several investigators attempted to correlate the stage of maturation of the male gamete with its reproductive capacity. It has been shown that the more advanced the round spermatid stage is, the larger its reproductive capacity is (Sofikitis et al.., 1997). However, it should be emphasized that the nucleus of the male gamete even in very immature stages (i.e., round spermatid prior to appearance of acrosomal granule, secodary spermatocyte, or primary spermatocyte) has capacity to fertilize oocytes (Kimura and Yanagimachi, 1995; Sasagawa et al. 1998). Goto et al. (1996) examined the possiblility of utilizing in vitro derived spermatids for intracytoplasmic injection. It was found that bovine oocytes injected with in vitro derived spermatids were capable of developing to blastocyst stage. CLINICAL APPLICATION OF OOPLASMIC INJECTIONS OF ROUND SPERMATIDS After the encouraging message from the above animal investigations an attractive challenge was to apply ooplasmic injections of spermatid nuclei selected from testicular biopsy material or the ejaculate for the treatment of non-obstructed azoospermic men (Sofikitis et al.,1995; Hannay, 1995). The first pregnancies in the international literature via ooplasmic injections of round spermatid nuclei were achieved in 1994 and reported in 1995 (Sofikitis et al., 1995a; Hannay, 1995). However, these pregnancies resulted in abortions. A few months later, Tesarik et al. (1995) reported delivery of two healthy children after round spermatid injections into oocytes. The mean fertilization rate was 45% in that study. Additional human pregnancies after ooplasmic injections of intact round spermatids or round spermatid nuclei were reported by Mansour et al. (1996), Tanaka et al. (1996), Antinori et al. (1997a and 1997b), Vanderzwalmen et al. (1997), Sofikitis et al. (1998a), and Barak et al., (1998). T h e first attempts to treat non-obstructive azoospermia with ooplasmic injections of round spermatids in the USA were performed in California, Louisiana, and Florida (Sofikitis et al., 1995 b ).Fertilization and development rate up to 10 cell stage embryos was achieved. The overall fertilizations rate per injected oocyte was 31% in that study. In addition, Fishel et al. (1995 and 1996), Amer et al. (1997), Barak et al., (1998) and Sofikitis et al. (1998b) reported human pregnancies after ooplasmic injections of elongating or elongated spermatids. The knowledge gained from all the above studies increased our understanding on the reproductive potential of the early haploid male gamete and was translated into successful application of ooplasmic injections of human secondary spermatocytes (Sofikitis et al. 1998c). Delivery of a healthy newborn was reported in that study. Amer et al. (1 997) u sed the term "complete spermiogenesis failure" for non-obstructed azoospermic men in whom the most advanced spermatogenic cell present in testicular biopsy material is the round spermatid and the term "incomplete spermiogenesis failure" for non-obstructed azoospermic men with a very limited number of elongated spermatids in testicular biopsy material. Several studies have shown clearly that in men with spermatogenic arrest at the primary spermatocyte stage or Sertoli cell-only syndrome a number of germ cells in a limited number of tubules can break the barrier of the premeiotic spermatogenic block and differentiate up to the stage of round spermatid (Mansour et al., 1996;) Tanaka et al. 1996; Antinori et al. 1997a; Vanderzwalmet et al, 1997; Sofikitis et al. 1998a). Maturation arrest at the round spermatid stage may be due to acquired primary or secondary testicular damage (Sofikitis et al., 1998a) or genetic factors (Weinbauer et al., 1998). In addition complete maturation arrest can be produced in experimental animals by deficiency of hormones and other regulatory factors, as well as by targeted mutation of genes for receptors, elements for signal transduction pathways, or intracellular repair systems (Tesarik, 1998). A diagnostic testicular b iopsy negative for round spermatids does not rule out the probability that few or many round spermatids will be found in the therapeutic testicular biopsy material. Furthermore, peripheral serum levels of FSH and testicular size do not predict the presence or absence of round spermatids in the therapeutic testicular biopsy material. It should be emphasized that the diagnostic testicular biopsy refers to the evaluation of a limited amount of tissue recovered from one testicular location. Diagnostic testicular biopsy material is exposed to various detergents during fixation/staining and subsequently a number of cells are degenerated and their identity cannot be defined. In contrast, therapeutic testicular biopsy refers to the isolation of a larger amount of tissue from several areas of testicular

11 Treatment of Non-Obstructive Azoospermia with Ooplasmic Injections of Immature Germ Cells 11 tissue. Therapeutic testicular biopsy specimens are minced for hours into isoosmotic solutions containing energy sources and isolated, single cells can be observed. Therefore, the value of the diagnostic testicular biopsy in the management of non-obstructed azoospermic men should be questioned. Combined analysis of our studies in the management of nonobstructed azoospermic men (Sofikitis et al., 1995a; Sofikitis et al.,1995b; Sofikitis et al., 1998a;Sofikitis et al., 1998b; Yamanaka et al., 1997) showed that among the men who participate for first time in assisted reproduction programs (regardless of positive or negative results for spermatozoa in the diagnostic testicular biopsy), a percentage of 39% have spermatozoa in their therapeutic testicular biopsy material. For a percentage of 43% the most advanced spermatogenic cells are spermatids in the theraputic testicular biopsy s p e c i m e n (Sofikitis et al., 1998a). These men are candidates for ooplasmic injections of round spermatids or round spermatid n uclei. It appears that no wadays a small percentage only of non-obstructed azoospermic men (18% approximately) are excluded from assisted reproductive techniques. CONTRIBUTIONS OF THE ROUND SPERMATID TO ZYGOTE Th e male gamete contribu tes several components important for the fertilization process and early embryonic development to th e zygote: the genetic material, the reproducing element of the centrosome, the microtubule organizing component of centrosome, the oocyte-activating factor (s), nuclear proteins, and factors affecting early embryonic development and capacity for implantation (see for review Sofikitis et al.,1998a). The deliveries of healthy animal offspring and healthy human newborns after ooplasmic injections of round spermatids indicate that the above mentioned components of the early haploid male gamete are mature and efficient to initiate/participate in the cascade of ooplasmic events that lead to completion of the fertilization process. However, probable inefficiency of some of the male gamete components to accomplish their missions may contribute to the low outcome of ooplasmic injection of round spermatids. Genetic material The deliveries of normal mouse and rabbit offspring and healthy human newborns after ooplasmic injections of round spermatids indicate the maturity of the genetic material of the early haploid male gamete and suggest that the chromosomes of the round spermatid are capable of pairing with those of the oocyte and participate in syngamy, fertilization, and subsequent embryonic and fetal development. Oocyte activation factor The male gamete induced cascade of biochemical ooplasmic events that results in resumption of meiosis of the female gamete is referred to as oocyte activation. Oocyte activation is a prerequisite for male pronucleus formation and subsequently for fertilization. Therefore, an anatomical or functional defect of oocyte-activating factor may affect dertimentally the fertilization process after ooplasmic injections of round spermatids. Ooplasmic injections of mouse round spermatids do not result in oocyte activation suggesting that the mouse oocyteactivating factor has not been expressed at the round spermatid stage (Kimura and Yanagimachi, 1995). In contrast, ooplasmic injections of rabbit round spermatids lead to oocyte activation in a significant percentage (Sofikitis et al., 1994). Electrical stimulation of the rabbit oocyte enhances the oocyte-activating factor and benefits the activation process (Sofikitis et al., 1996a). Although electrical stimulation usually results in a monophasic ooplasmic Ca 2+ response, it appears that there is a synergistic action of electrical stimulation and round spermatid oocyte-activating factor which eventually produces Ca + 2 oscillations and results in oocyte activation. There is strong evidence that the human oocyte-activating factor is expressed at/before the round spermatid stage (Yamanaka et al., 1997; Sousa et al.,1996). The achievement of human pregnacies via ooplasmic injections of round spermatids without application of an exogenous electrical or chemical stimulation supports the above thesis. The human oocyte activatio n after ooplasmic injections of round spermatids may not be attributed to parthenogenetic activation of the oocytes since ooplasmic injections of medium only have not resulted in activation (Fishel et al., 1996 ; Yamanaka et al., 1997). Meng and Wolf (1997) h a v e emphasized that human or monkey mechanical ooplasmic stimulation and/or exposure to a low or a relatively high extracellular calcium concentration of medium can alter intracellular Ca + 2 but, not alone, cause activation. Recent studies have suggested that there may be a deficiency of ooyte-activating factor in a subpopulation of non-obstructed azoospermic men (Sofikitis et al., 1998a). In the latter men application of electrical or chemical stimulation prior, during, or im mediately after ooplasmic injection s of roun d spermatids may act synergetically with the ooclyte-activating factor present in the round spermatid and increase activation, fertilization and pregnancy rates. Centrosomic components of the round spermatid. The zygote s centrosome is a mixture of paternal and maternal components. T he restoration of th e zygotic centrosome at fertilization requires the attraction of maternal centrosomal components to the paternal reproducing

12 12 Nikolaos Sofikitis element of the centrosome (Schatten, 1994). The male gamete contributes to the zygotic centrosome by transferring the reproducing element of the centrosome, the microtubule organizing center, and a 7 tubulin binding protein (Schatten, 1994). The delivery of healty babies after human ooplasmic injections of round spermatids tends to suggest that the centrosomic components of the human round spermatid are normal, functional, and mature. Centrosomic abnormalities in round spermatids may result in aberrant spindle formation and subsequently in an increased risk of mosaicism. Nuclear proteins Following ooplasmic injections of round spermatids or round spermatid nuclei and disintegration of the round spermatid nuclear membrane within the ooplasm, the round spermatid DNA-nuclear protein comlex is exposed to ooplasmic factors. Since the round spermatid nuclear proteins are histones (Perreault and Zirkin, 1982) known to contain a reduced number of disulphide bonds, a question may be raised as to how the round spermatid DNA that is not associated with disulphide bond-proteins can survive within the ooplasm. Lack of protection of the round spermatid DNA may be responsible for the premature irreversible condensation of the chromosomes of the round spermatid frequently demonstrated after ooplasmic injections. The latter may contribute to the smaller fertilization rates after ooplasmic injections of round spermatids comparatively with ooplasmic ingections of spermatozoa. Factors affecting early embryonic development Janny and Menezo (1994) have provided strong evidence that the mission of the male gamete is not only to activate and fertilize the oocyte but also to contribute to the zygote potential to undergo the first mitotic divisions. It appears that there is a paternal effect on early embryonic development. Sofikitis et al. (1996b) showed a defect in the capacity for early development and implantation of embryos generated from the fertilization of oocytes by round spermatids. The lower potential for development of embryos derived from the fertilization of oocytes with round spermatids may be attributable to a deficiency of the round spermatid factors mediating th e paternal influence on the embryonic development. GUIDELINES FOR OOPLASMIC INJECTIONS OF HUMAN ROUND SPERMATIDS Quality control for identification of round spermatids The gold standard for identification of round spermatids is transmission electron microscopy. Another valid approach is fluorescent in-situ hybridization. A drawbach to the above techniques is that application of these methods results in cell death. Therefore, observed spermatids cannot be used in assisted reproduction programs. Indeed one of the most perplexing problems in the laboratories applying ooplasmic injections of human round spermatids is the identification of alive, undisturbed, non-fixed, non-stained round spermatids. Quantitative and qualitative criteria have been proposed for indentification of undisturbed round spermatids (Sofikitis et al., 1998a). Training is necessary for the staff of assisted reproduction centers applying ooplasmic injections of round spermatids. Technicians.embryologists/physicians performing ooplasmic injections of round spermatids should also confirm via transission electron microscopy or fluorescent in-situ hybridization techniques that the cells that are considered as round spermatids are indeed round spermatids. Quality control for viability of round spermatids Fractions of round spermatids retrieved from testicular tissue should be processed for assessment of viability. Men with percentage of live round spermatids <10% have a poor fertilization outcome after ooplasmic injections (Sofikitis et al., 1998a). Quality control for the capacity of round spermatids to activate oocytes. A previous study has shown that failure of ooplasmic injections of round spermatids may be attributable to subnormal profiles/activity of the oocyte-activating factor. Application of a recently/reported quantitative assay to appreciate oocyte-activating factor activity is recommended (Sofikitis et al., 1998a; two round spermatids are injected into a hamster oocyte). If the percentage of activated hamster oocytes in the latter assay is <8%, fertilization is not anticipated after human ooplasmic injections of round spermatids. Stage of the round spermatid and fertilization Round spermatids with the larger acrosomal bright/dark spots (observation via inverted microscopy) should be processed for ooplasmic injections because they represent the most mature of the early haploid male gamete. We have shown that round spermatids of steps 1 and 2 have smaller reproductive potential than round spermatids of stage 3-5 (Sofikitis et al, 1997). Media for maintenance of round spermatids Recently, specific mediums have been developed for maintenance of round spermatids (Yamanaka et al., 1997), These madiums contain lactate and glucose as energy substrates. Lactate is the preferable enery substrate for round spermatids. Round spermatids have a larger amount of cytoplasm than spermatozoa. To protect spermatids against environ men tal shock and to stabilize the spermatid

13 Treatment of Non-Obstructive Azoospermia with Ooplasmic Injections of Immature Germ Cells 13 membrane, cholesterol has been added to mediums used for maintence of round spermatids. Media for culture of oocytes injected with round spermatids Several studies have shown that the addition of antioxidants to media used for culture of embryos generated from fertilization of oocytes with spermatids has beneficial effects on embryonic development (Sofikitis et al. 1996a; Sofikitis et al., 1996b). GENETIC RISKS AFTER OOPLASMIC INJECTIONS OF ROUND SPERMATIDS To evaluate the genetic risks of assisted reproductive technologies one has to consider the genetic risks inherent to the treatment population and the genetic risks inherent to the procedure performed. Genetic risks inherent to ooplasmic injections of immature germ cells may involve injection of disomic/diploid genetic material which could give rise at fertilization to a trisomic/triploid embryo and fetus, genomic imprinting abnormalities, and abnormalities due to the out-of phase cycles of the round spermatid (G1 stage) and the oocyte (M phase). Genetic risks inherent to a population of men with primary testicular damage are the same with the genetic risks of ooplasmic injections of spermatozoa (transferring sex chromosomal abnormallities or reciprocal translocations associated with spermatogenic impairment). Inheritance of gene mutations/deletions of DNA sequences of specific regions of the Y- chromosome long arm represent additional risks. Taking into consideration that abnormalities in genomic imprinting are associated with genetic diseases, a question of great clinical importance is whether genomic imprinting has been completed at the human round spermatid stage. To attempt to answer this question the imprinting of a gene should be divided into three stages: erasure of the previous imprint, re-imprinting, and consolidation of the new reprint. There is strong evidence that erasure of the previous imprint occurs prior to meiosis and that re-establishment of the new imprint begins prior to the pachytene stage of meiosis (Tycko, ). In contrast, the fact that DNA methyltransferase enzyme is present in spermatids may be an argument against the thesis that genomic imprinting is complete at the round spermatid stage. However, waves of DNA methylation have been demonstrated during early embryonic development, the blastocyst stage, and the time of implantation (Fishel et al., ). These observations tend to suggest that even if genomic imprinting is not complete at the round spermatid stage, genomic imprinting may be completed after the transfer of the round spermatid within the ooplasm. REFERENCES 1. Fawcet D, Anderson W, Phillips D (1971): Morphogenetic factors influencing the shape of the sperm head. Dev Biol, 26, Perreault S, Zirkin B (1982): Sperm nuclear decondesation in nammlas: role of sperm associated proteinase in vivo. J Exp Zool, 224, Ogura A, Yanagimachi R (1993): Round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. Biol Reprod, OguraA, Yanagimachi R, Usui N (1993): Behavior of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion. Zygote, 1, Ogura A, Matsuda J, Yanagimachi R (1994): Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc Natl Acad Sci USA, Sofikitis N, Miyagawa I, Agapitos E et al. (1994): Reproductive potential of the nucleus of the male gamete after completion of meiosis. J Asist reprod Genet, 11, Sofikitis N, Toda T, Miyagawa I et al. (1996a): Beneficial effects of electrical stimulation before round spermatid nuclei injections into rabbit oocytes on fertilization and subsequent embryonic devepopment. Fertil Steril, 65, Sofikitis N, Miyagawa I, Incze P et al. (1996b): Detrimental effect of left varicocele on the reproductive potential of early haploid male gamete. I Urol, 156, Sasagawa I, Yanagimachi R(1997): Spermatid from mice after crytorchid and reversal operations can initiate normal embryo development. J Androl, 18, Sofikitis N, Yamamoto Y, Miyagawa I(1997): The early haploid male gamete develops a capacity for fertilization after the coalescence of the proacrosomal granules. Hum Reprod, 12, Kimura Y, Yanagimachi R (1995): Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biol Reprod, 53, Sasagawa I, Kuretake S, Epping J, Yanagimachi R (1998): Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm. Biol Reprod, 58, Goto K, Kinoshita A, Nakanishi Y (1996): Blastocyst formation following intracytoplasmic injection of in vitro derived spermatids into bovine oocytes. Hum Reprod, 11, Hannay T(1995): New Japanese IVF method finally made available in Japan.Nature Medicine, 1, Sofikitis N, Miyagawa I, Sharlip I et al. (1995a): H u m a n pregnancies achieved by intraooplasmic injections of round spermatid nuclei isolated from testicular tissue of non-obstructed azoospermic men. J Urol, 153 (Suppl), 258A. 16. Tesarik J, Mendoza C, Testart J (19995): Viable embryos from injections of round spermatids into oocytes. N Engl J Med, 333, Mansour R, Aboulghar M, Serour G, (1996): Pregnancy and delivery after ooplasmic injection of spermatids into human oocytes. Middle East Fertil Soc, 1,

14 14 Nikolaos Sofikitis 18. Tanaka A, Nagayoshi M, Awata S (1996): Clinical evaluation of round spermatid injection into human oocytes. Fertil Steril, 66(Suppl), p S Antinori S, Versaci C, Dani G et al. (1997a): Fertilization with human testicular spermatids: four successful pregnancies. Hum Reprod, 12, Antinori S, Versaci C, Dani G et al. (1997b): S u c c e s s f u l fertilization and pregnancy after injections of frozen-thawed round spermatids into human oocytes. Hum Reprod, 12, Vanderzwalmen P, Zech H, Birkenfeld P et al. (1997): Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids, and oocyte activation. Hum Reprod, 12, Sofikitis N, Miyagawa I, Yamamoto Y et al. (1998a): Micro- and macro-consequences of ooplasmic injections of early haploid male gametes. Hum Reprod Update, 4, Barak Y, Kogosowski A, Goldman S et al. (1998): P r e g n a n c y and birth after transfer of embryos that developed from singlenucleated zygotes obtained by injection of round spermatids into oocytes. Fertil Steril 70, Sofikitis N, Toda T, Miyagawa I et al. (1995b): Application of ooplasmic round spermatid nuclear injections for the treatment of non-obstructive azoospermia in USA. Fertil Steril 64 (Suppl), S88- S Fishel S, Green S, Bishop M et al. (1995): Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, Fishel S, Aslam I, Tesarik J (1996): Spermatid conception: a stage too early, or a time too soon? Hum Reprod, 11, Amer M, Soliman E, El-Sadek M et al. (1997): Is complete spermiogenesis failure a good indication for spermatid conception? Lancet, 350, Sofikitis N, Yamamoto Y, Miyagawa I (1998b): O o p l a s m i c elongating spermatid injections for the treatment of nonobstructive azoospermia. Hum Reprod, 13, Sofikitis N, Mantzavinos T, Loutradis D (1998c): O o p l a s m i c injections of secondary spermatocytes for non-obstructive azoospermia. Lancet, 351, Weinbauer G, Behr R, Bergman M et al. (1998): Testicular camp responsive element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest. Mol Hum Reprod, 4, Tesarik J (1998): Spermatid conception. Hum Reprod, 13: Yamanaka K, Sofikitis N, Miyagawa I et al. (1997): O o p l a s m i c round spermatid nuclear injections as an experimental treatment for non-obstructive azoospermia. J Assist Reprod Genes, 14, Sousa M, Barros A, Tesarik J (1996): Calcim responses of human oocytes after intracytoplasmic injection of leukocytes, spermatocytes, and round spermatids. Mol Hum Reprod, 2, Meng L, Wolf D(1997): Sperm induced oocyte activation in the rhesus monkey: nuclear and cytoplasmic changes following intracytoplasmic sperm injections. Hum Reprod, 12, Schatten G (1994): The centrosome and its mode of inheritance: the reduction of centrosome during gametogenesis and its restoration during fertilization. Dev Biol, 165, Janny L, Menezo Y (1994): Evidence for strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol Reprod Dev, 38, Tycko B (1997): Post-graduate course program of The American Society of Andrology, 22nd Annual Meeting, Baltimore, Maryland, February 22-25, pp

15 AÓ Ú, (3), AºIEPøMA MEPO 2Æ RECENT ADVANCES AND UPDATE IN ASSISTED REPRODUCTIVE TECHNOLOGIES P.M. ZAVOS, Ed.S., Ph.D., Professor REPRODUCTIVE PHYSIOLOGY & ANDROLOGY, UNIVERSITY OF KENTUCKY, LEXINGTON, KY, USA DIRECTOR, ANDROLOGY INSTITUTE OF AMERICA, LEXINGTON, KY 40523, USA ASSOCIATE DIRECTOR, KENTUCKY CENTER FOR REPRODUCTIVE MEDICINE & IVF, LEXINGTON, KY INTRODUCTION Employment of IVF technologies since their introduction in 1978 brought about a revolution in the mode of treatment of human infertility in general. Meanwh ile, since the development of the intracytoplasmic sperm injection (ICSI) in 1992, this technique is now widely applied in couples with severe male factor infertility. Using this method, we have gathered valuable information regarding the dynamics of the interactions of the male and female gametes during the fertilization process. For those patients that lack sperm in the ejaculate, microsurgical methods of sperm retrieval from the testis (TESE) or epididymis were introduced, which in combination with ICSI, yield adequate fertilization and pregnancy rates. However, in severe cases, when no sperm could be found in the testis or epididymis, the retrieval and injection of round spermatids (ROSI) into the oocyte via ICSI has been shown to be a viable alternative. Round spermatids contain a complete haploid set of chromosomes and when injected into human oocytes form pronuclei and participate in syngamy yielding full pregnancies. In cases of failed implantation, the process of assisted hatching has received widespread attention. This method creates a small hole, a thinning or a total removal of the zona pellucida, and has been shown to increase pregnancy rates in patients of older age, or with elevated basal FSH, or embryos with thickened zonae pellucidae. On a different level, embryos are routinely transferred on day 2 or 3 post fertilization (4-8 cell stage). The current trend is shifting towards culturing embryos to the blastocyst stage and transferring them on day 5. The main concern, however, is to develop consistent, safe and effective culture systems to obtain an adequate percentage of embryos and/or blastocysts that whenever transferred, could yield a viable pregnancy. This review will address the advantages and disadvantages of the latest developments in Assisted Reproductive Technologies (ART), and how they should be applied. SEMEN COLLECTION The proper evaluation of semen is certainly one of the most important factors in the investigation of an infertile couple. Harvesting the maximum quantity and quality of spermatozoa is of extreme importance in an artificial insemination (AI) program, or other forms of Assisted Reproductive Technologies (ART), using husband (AIH) or donor (AID) semen for infertility treatment purposes ( 1 ). Semen specimens collected for evaluation should resemble the ejaculate delivered during intercourse, as closely as possible, if the male infertility factor is to be properly identified and treated (2-9). Seminal characteristics in the

16 16 P. M. Zavos various animal species can be influenced by factors such as the frequency of semen collection, extent of precoital sexual stimulation (PSS) and degree of stabilization of epididymal sperm reserves ( 1 0 ). Meanwhile, as of today, the most accepted method of semen collection in humans for the purpose of semen analysis, AI or ART is via masturbation (2-9). Recent developments have allowed semen collection to be performed via the use of semen collection devices (SCD). Those devices generally consist of nonspermicidal condoms made of polyurethane or silicone rubber (2, 3, 5, 7-9, 11, 12). Due to their acceptability by patients, lack of negative effects on sperm viability, and assistance in the improvement of collected specimens to closely resemble the ejaculates obtained at intercourse, these devices may be used routinely for semen collection for use in the various ART and intrauterine insemination (IUI) procedures (8, 9, 11, 13-17). It has also been previously established that by employing the SCD method, the male is generally better able to achieve a greater degree of sexual stimulation prior to ejaculation and production of a seminal specimen (8, 9, 11,15). We have systematically sho wn along with others that seminal specimens obtained at intercourse via the use of SCD s exhibited greater seminal plasma volume, total sperm count, motility characteristics, and normal morphological features than those collected via masturbation ( 2-9 ). In an additional study, the markers of the secretory function of the prostate and the outcome of various sperm functional tests such as the hypoosmotic swelling (HOS) test, acrosin, and sperm penetration assays, were significantly higher for sperm samples collected at intercourse than for those collected via masturbation ( 1 8 ). The findings in a more recent study indicated that ejaculates produced via the complete coitus method are of larger volume and contain larger numbers of spermatozoa with greater motility than those compared with ejaculates obtained via the coitus interruptus method, although the sperm density per ml did not differ between the two semen collection methods ( 1 5 ). It was suggested that during the production of ejaculates via the complete coitus method, the apparent sexual stimulation of the males by their sexual partners during the later part of ejaculation process was actively involved in the completion of the ejaculatory process, which also could have brought about the observed ejaculate improvements (15). We have also demonstrated that ejaculates produced under increasing intensity of precoital sexual stimulation (PSS) can bring about significant increases in ejaculate characteristics (Table 1). Although the direct cause of such stimulation is not completely understood, it can be suggested that PSS may be responsible for the greater loading of the vas deferens during coital sexual stimulation and subsequent unloading of the vas deferens during the duration of the ejaculatory process achieved during the longer stimulatory period (9, 19). Also, precoital sexual stimulation (PSS) results in increases in seminal characteristics, and should be considered during productio n o f seminal specimens, especially in patients with spermatogenic dysfunctions such as hypospermia, oligospermia, asthenospermia or others. These improved sperm specimens could then be used further to improve fertilization and pregnancy results in the various assisted reproductive technologies, improve the accuracy of male infertility diagnosis or to maximize the sperm harvest during semen preparation. Table 1. Seminal parameters of ejaculates collected via the three different degrees of precoital sexual stimulation (PSS; means±sd) Seminal characteristics assessed Degree Volume Count Motility Grade Morphology Longevity of PSS* (ml) (x10 6 ) (%) (0 to 4) (% normal) (% motility) Mode A 5.3±1.1 a 237.8±23.4 a 63.4±7.3 a 3.6±0.2 a 58.6±6.7 a 38.7±5.1 a Mode B 3.6±0.9 b 136.4±18.7 b 55.1±6.1 b 3.2±0.2 a 51.0±6.5 a,b 24.3±6.1 b Mode C 3.1±1.2 b 110.1±15.6 b 50.3±6.3 b 2.9±0.3 b 46.7±6.0 b 20.9±5.0 b *Mode A consisted of attempting intercourse until before the time of ejaculation and stopping at this time (Attempt 1). Patients were instructed to repeat the procedure after 12 hours (Attempt 2), followed by entering a 5-minute resting (refractory) period and then trying again to complete ejaculation (Attempt 3). Mode B consisted of producing an ejaculate as in Mode A by attempting incomplete intercourse, followed by a 5 minute resting period and reattempting complete intercourse. Mode C consisted of producing an ejaculate at the first attempt. Longevity was measured as the percentage of motility after 72 hours of incubation in humidified 5% CO2 in air at 37ÆC. a,b Values with the different superscripts are significantly different (P<0.05). SEMEN PREPARATION One of the most important steps prior to the performance of intrauterine insemination (IUI), in-vitro fertilization (IVF) or other forms of Assisted Reproductive Technologies, is to remove the seminal plasma from the ejaculate and harvest the mo tile fraction to ensure successful fertilization. Therefore, the goal of any sperm separation procedure is to retrieve a maximum number of viable sperm whose biological functions are maintained by means of the separation technique. This is mandatory not only for treatment cycles in

17 Recent Advances and Update in Assisted Reproductive Technologies 17 assisted reproduction, but also for the assessment of spermatozoa functions which should be performed in the diagnostic work-up of male infertility. Techniques for the preparation of sperm include a simple wash with centrifugation and mechanical resuspension ( 2 0 ), Percoll gradients ( 2 1 ), swim-up procedures ( 2 2 ), albumin gradients ( 2 3 ), glass wool filtration ( 2 4 ), Sephadex columns ( 2 5 ), Ficoll columns ( 2 6 ) and migration sedimentation ( 2 7 ). The methods share the common goal of recovery of sperm from seminal fluid with the highest possible yield, producing a specimen of high count and motility that is free of seminal proteins and microbial contamination, without introducing iatrogenic effects that may diminish sperm motility, viability and, ultimately, fertilizing potential. The goals of sperm preparation for IVF and IUI differ. While lower numbers of sperm may be acceptable at the cost of higher quality for IVF, in the case of sperm preparation for IUI, it is considered necessary to have a suspension containing as many high quality sperm as possible. Recent developments have focused on semen preparation methods more critically especially since the withdrawal of Percoll from the market. Various other products, though similar in composition to Percoll have been introduced, with lower endotoxin levels and have shown to be effective in isolating motile sperm from the specimen. However, it has been suggested that with the increased number of motile sperm retrieved via this method, the fertilizing ability of the sperm may be compromised since morphologically abnormal, yet motile, sperm are recovered. In addition, the long term effects of these colloidal particles are not known. Also, concerns have focused on the possibility of mechanical damage to sperm from centrifugation and resuspension, leucocyte and bacterial contamination and contamination of the sperm sample with elements from the preparation medium. Centrifugation-induced mechanical damage in particular may lead to low recovery of motile sperm, may damage sperm integrity and increase levels of reactive oxygen species (ROS), particularly in specimens with poor semen characteristics (28). In order to prevent any centrifugal damage and/or compromise to sperm quality, it has been suggested that sperm preparation should utilize the natural migration of sperm out of the semen via their own innate motility. In this manner, one can ensure that these sperm are functionally and genetically superior to those that do not swim-up. Zavos et al ( 2 9 ) have introduced a modified swim-up technique that utilizes the swim up/swim down migration of sperm out of the seminal plasma into the culture medium. The Zavos Swim-up Column (ZSC) (ZDL, Inc, Lexington, KY, USA), is a simple self-contained system for the recovery of high-quality progressively motile sperm from semen. The Sperm Medium Semen Source Figure 1. Diagrammatic illustration of the ZSC device, showing motile sperm swimming up from the semen into the medium Chamber 4 Chamber 3 Chamber 2 Chamber 1 Semen Source Figure 2. Diagrammatic illustration of the Multi-ZSC device, showing the orientation of the various chambers above the semen source filled with sperm medium. ZSC has been reported to be a fast, inexpensive and simple one-step standardized method. The ZSC washes and simultaneously selects sperm, thus eliminating the need for dilution and centrifugation. The ZSC column combines the swim-up/swim-down methods, yielding a greater number of high-quality, morphologically normal, motile sperm. Because the semen specimen is safely protected within the conical cavity inside the ZSC column prior to the addition of medium, the risks of mixing the medium with the specimen are eliminated. Thus, the possible contamination of the medium that contains the healthy sperm is totally reduced. The incubation period (15 minutes to one hour depending upon the pro cedure to be performed) causes motile and morphologically normal sperm to swim up and out of the cone, and down into medium. When the ZSC column is used in conjunction with SpermPrep medium, maximal retrieval of highly motile sperm can be expected.

18 18 P. M. Zavos The ZSC procedure was compared to the SpermSelect system and both devices yielded sperm of similar qualitative characteristics (29). Table 2. Characteristics of sperm obtained in the different chambers of the Multi-ZSC using normozoospermic samples Normozoospermic samples (n=30) Characteristics Raw Ch-1 Ch-2 Ch-3 Ch-4 Count (x10 6 ) 117±6.8* 21.0± ± ± ±1.4 Motility % 61.0±0.5* 88.5± ± ± ±0 Grade (%) 29.2±1.1* 81.0± ± ± ±0 Morphology(%)35.0±1.3* 48.6± ± ± ±1.2 *Significant differences (P<0.05) vs subpopulations of spermatozoa harvested from the swim-up column. Table 3. Characteristics of sperm obtained in the different chambers of the Multi-ZSC using teratozoospermic samples Teratozoospermic samples (n=30) Characteristics Raw Ch-1 Ch-2 Ch-3 Ch-4 Count (x10 6 ) 179.0±14.7* 33.2± ± ± ±0.3 Motility % 55.1±1.8* 84.7± ±0 99.8± ±0 Grade (%) 27.1±2.5* 78.0± ±0 99.8± ±0 Morphology(%)17.5±1.5* 41.3± ± ± ±2.5 *Significant differences (P<0.05) vs subpopulations of spermatozoa harvested from the swim-up column. Table 4: Preparation and technologist times involved in sperm preparation methods Method Total time Technologist time Percoll 40 minutes 12 minutes Iodixanol 54 minutes 12 minutes Accudenz 40 minutes 12 minutes Nycodenz 40 minutes 12 minutes SpermPrep II 54 minutes 13 minutes Zavos Swim-up minutes 5 minutes Swim-up 90 minutes 10 minutes Glass wool filtration 25 minutes 13 minutes Centrifuge and resuspension 25 minutes 10 minutes Transmembrane methods 20 minutes 5 minutes *Taken from Davies and Cumming (33) INTRACYTOPLASMIC SPERM INJECTION (ICSI) AND ROUND SPEMATID INJECTION (ROSI) Intracytoplasmic sperm injection (ICSI) has been indicated in cases of previously failed fertilization or severe male factor infertility. This method has been applied extensively and has shown great success. However, there are concerns as to whether this method should be applied in all cases of infertility and where ART s are indicated. Various innovations and improvements to the ICSI method itself have been developed and implemented. These have focused on the selection and capture of viable sperm for injection, rupture of the oolemma, and the breaking of the tail. It is established now that the sperm tail should be broken prior to injection. The rationale for this is yet unclear. For cases of 100% immotile sperm, the placement of sperm into a hypoosmotic solution (±150mOsm) in order to select viable sperm based on the coiling of the tail has been used. This method has proven to be successful and eliminated the need for invasive methods of sperm retrieval such as testicular sperm extraction (TESE) or microsurgical sperm aspiration (MESA). The rupture of the oolemma still seems to be one of the crucial steps to fertilization success during ICSI. The most common means to achieve this is prior to the injection of the sperm, to aspirate slowly until the ooplasm enters the injection pipette, thereby ensuring that the oolemma has been broken. This step is crucial since it allows proper placement of the sperm into the ooplasm and also initiates a whole cascade of events leading to oocyte activation and proper fertilization. In cases of obstructive azoospermia, the use of testicular or epididymal sperm has been shown to result in fertilization and subsequent pregnancies. Typically, a piece of testicular tissue would be excised, the seminiferous tubules dissected and sperm recovered in the surrounding media. Alternatively, sperm could be aspirated directly from the epididymis, which in most cases yield a much cleaner specimen, with motile spermatozoa. Recent efforts in the area of male reproduction and treatment of male infertility have employed the use of round (RS) and elongated (ES) spermatids utilizing intra cytoplasmic sperm injection (ICSI) techniques for the purpose of achieving pregnancies. Such efforts have yielded relatively satisfactory fertilization rates but extremely low pregnancy results. In most instances the immature spermatozoa (RS and ES) are recovered via testicular biopsies and in a few instances from post-ejaculated semen (non-obstructive azoospermia). Meanwhile, the pregnancy results from such recoveries have been somewhat conflicting although not adequate numbers exist to draw significant conclusions.

19 Recent Advances and Update in Assisted Reproductive Technologies 19 In the absence of spermatozoa following TESE or MESA, the injection of round spermatids (ROSI) have led to considerable debate to its efficacy. Despite reports of successful fertilization and pregnancies using this method, there exists great discrepancy in the selection of viable round spermatids, which could result in poor results. Several pregnancies have been achieved via intracytoplasmic injection of round spermatids or round spermatid nuclei (ROSNI). Furthermore, the injection of elongated spermatids have also resulted in human pregnancies. However, it has also been reported that injection of testicular round spermatids resulted in lower fertilization rate and a higher incidence of development arrest in embryos compared to injection of mature sperm recovered from the testes. In cases of non-obstructive azoospermia, rou nd spermatids can be found in the ejaculate, thereby possibly eliminating th e need for invasive procedures such as testicular biopsy via needle aspiration techniques or open biopsy methods. In one study, we have compared the morphometric characteristics of round spermatids obtained from the testes with those from the ejaculate (34). The data from that study indicated that the overall size of the round spermatid was similar between the two sources of the spermatids (Table 5). However, there were significant Table 5. Size and surface areas of the various compartments of round spermatids recovered at post-ejaculation and from the testes via testicular biopsy. Spermatid compartments Round Spermatid Source Ejaculated (N=8) Non-Ejaculated (N=10) Spermatid Size (Ìm) 8.2± ±0.2 Cytoplasm (%) ±1.2a 59.3±4.1 Nucleus (%) ±0.8a 35.7±0.9 Acrosome (%) 1 8.1±0.2 a 6.0±1.3 Homogeneity (%) 94.3 a 62.7 capacity of these spermatids remains to be investigated. One of the most perplexing problems in the laboratories applying intracytoplasmic injections of h uman rou nd spermatids is the identification of live, undisturbed, non-fixed, non-stained round spermatids. We have recently introduced a simplified method for the identification and isolation of viable round spermatids that could be used for ICSI ( 3 5 ). This method involves the placement of RS in a hyper-osmotic solution which will cause the cell to shrink and can be identified as a spermatid with an intact membrane that when used in ICSI could yield complete fertilization. We have addressed the importance of distinguishing and selecting high quality, viable RS from other RS, by employing techniques that have been previously described (36, 37). Also, the presence of debris is an important aspect which could interfere with the performance of the R OSI micromanipulatio n steps and especially during th e identification, pick-up and microinjection of the RS into the o o c y t e s ( 3 7 ). Round spermatids can be recognized by their size, form of nucleus, a defined cytoplasm surrounding the nucleus and the developing acrosome vesicle as noted in the current study and also in previous publications ( ). RS can also be differentiated from other cells by staining and labeling techniques (36). The applied purification techniques (37) enabled the proper selection of the round spermatids for proper testing and evaluation to take place. We have introduced a hyper osmotic shrinkage test (HYOS) which allows spermatids to shrink once placed into this medium (Figure 3). After the proper isolation, identification, purification (37, 39) and quality assessment of the RS via the HYOS-test, the injection of those cells into human oocytes should be relatively simple, since the technology and instrumentation for ROSI is quite similar to those methods cu rrently employed in ICSI, requiring o nly minute 1 Percentage distribution of total surface area of RS; a Significant difference between columns (P<0.05) differences in the percentage distribution of the various compartments of round spermatids obtained from the ejaculate compared to those recovered from the testes, in particular to the cytoplasmic and nuclear areas. However, the overall dimensions of the nuclear and acrosomal areas remained constant between the two spermatid sources. This would indicate a shift in the morphological characteristics of the spermatids as a result of maturation as they traverse the epididymis similar to those described for ejaculated spermatozoa. Whether these morphological differences noted between the two spermatid populations recovered via the two different methods have an impact on the fertilizing Figure 3. Round spermatids that either responded (left) or did not respond (right) to the HYOS-test (430x). The size and ratio of the subcellular compartments (cytosol:nucleus) were altered after the response to the HYOS-test. Also the "notching pattern" was evident to the HYOS-test reacted spermatids.

20 20 P. M. Zavos modifications. Furthermore, the ROSI technique with properly isolated, quality assessed via the HYOS-test and microinjected RS can be relatively successful, as a therapeutic option, in establishing pregnancies and most importantly, be extremely beneficial fo r patients with impaired spermatogenesis. A major and extremely important aspect in the performance of ROSI today is the development of proper techniques for isolation of intact, physiologically active RS, which is, in these authors opinion, one of the key elements for a successful ROSI program and a prerequisite for widespread use of ROSI as a new treatment for the nonobstructive and obstructive azoospermic patient. Different methods of assisted hatching include 1. Partial Zona Dissection (PZD) 2. Acidified Tyrodes / Pronase / Contact Laser 3. Non-contact Laser 4. Zona Thinning / Zona Removal These methods have not yet been scientifically compared. The actual day for assisted hatching varies between different investigators, where some prefer to use acidified Tyrodes on Day 3 (40) and others show better results using PZD on Day 2 (46). ASSISTED HATCHING After fertilization, the zona pellucida plays an important role in protecting the embryo during its transport in the fallopian tube. Thinning of the zona pellucida as the embryo develops is required for the proper hatching to occur. The hatching pro cess is a major event preceding embryo implantation. This phenomenon is mechanically related to the contraction and expansion of the blastocyst and probably also facilitated by the secretion of lysins of embryonic or uterine origin. Cohen et al (40) first suggested that a failure in the zona rupture could be involved in implantation failures. They have shown that embryos with either a thin zona pellucida or an artificial opening of the zona seemed to implant more frequently (41, 42). The absence or impairment of the hatching process could adversely affect implantation by preventing the contact between the trophoblast and the endometrium or by delaying the hatching process out of the implantation window (43). Assisted hatching consists of creating a thinning area or opening in the zona pellucida to facilitate the hatching process to take place. The indications for assisted hatching are: 1. Poor prognosis embryos, i.e. embryos with thick ZP (>13Ìm), low developmental rate, excessive fragmentation (43) 2. Patients of older age in which the zona could be thicker or harder. The age limit reported in different studies vary from 38 to 40 years old.(44) 3. Patients with elevated basal FSH levels.(44) 4. Patients with repeated failures of implantation 5. Frozen embryos, since hardening of the zona was reported after the freezing/thawing procedure.(45) SEQUENTIAL EMBRYO CULTURE AND BLASTOCYST TRANSFER Since the introduction of IVF, human preimplantation embryos are routinely transferred to the uterus around the 2 to 8-cell stage (on day 2 or 3). The current trend in humans is to transfer embryos at the blastocyst stage because this produces higher implantation and pregnancy rate ( ). However, the main concern with blastocyst transfer in humans is to develop consistent, safe and effective culture systems to obtain an adequate percentage of blastocysts. New culture conditions and media have been introduced to enhance embryo development in vitro. This is often termed sequential culture, which entails the placement of the embryo in different media based on the developmental stage of the embryo. Since the metabolic requirements of embryos change as they develop, it is thought that the culture media should accommodate for these changes. Day 1-2 Glucose-free and Phosphate-free Day 3 Low glucose and Phosphate-free Day 4-5 Phosphate free supplemented with amino acids Most programs, currently transfer embryos into Glucose and Phosphate free medium at the pronuclear stage and culture until time of transfer on Day 3. This protocol has resulted in the production of high quality embryos with minimal fragmentation, if any, and excellent pregnancy rates. The question of whether to transfer embryos at Day 2 or 3 stage versus blastocyst stage is probably the most controversial issue that confronts the ART community today. The potential advantages of blastocyst transfer (day 5) versus day 2/3 transfers are: 1. It is a more physiologically correct and appropriate approach in that the embryo enters the endometrial cavity

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