referring now to fig1 , a method 100 for embryo development after heterotopic transplantation of cryopreserved ovarian tissue will be explained . a woman was first diagnosed with cancer 102 or a collagen vascular , hematologic , or idiopathic disease requiring chemotherapy , radiotherapy , or a radical surgery that results in ovarian failure and infertility . ovarian tissue was collected via laparoscopy 104 or some other technique ( such as transvaginal or abdominal surgery ). in most cases , only one ovary will be removed , both to minimize trauma and because a single ovary provides a large amount of tissue . the ovary should be resected without the use electrosurgery , and the fallopian tube should be left intact so that the possibility of spontaneous pregnancy after future transplantation is maintained . once the ovary was removed , the ovarian cortex was sliced 106 into pieces ranging from 5 × 5 × 1 mm to 15 × 5 × 2 mm , although other sizes may also be used . these ovarian cortical pieces may be examined via biopsy prior to freezing to confirm that the ovarian tissue is free of any disease . these pieces were then cryopreserved 108 by placing the pieces into cryovials containing leibovitz l - 15 medium with 1 . 5 m 1 , 2 - propanediol , 20 % autologous serum , and 0 . 1 m sucrose . the cryovials were placed on a rotator and agitated for 30 minutes at 4 ° c . then the ovarian pieces were frozen using a slow - freeze protocol , in a programmable freezer . one technique for harvesting , cryopreserving , thawing , and transplanting ovarian tissue is disclosed in the book , “ a color atlas for human assisted reproduction .” see chapter fourteen , entitled “ the technique of ovarian transplantation : laboratory and clinical aspects ” by kutluk oktay and erkan buyuk (“ the technique of ovarian transplantation ”), which is herein incorporated by reference . after the ovarian tissue was preserved cryogenically , the cancer , collagen vascular , hematologic , or idiopathic disease was treated with chemotherapy , radiotherapy , or radical surgery 110 . once successful treatment is verified 112 , one vial of the ovarian cortical tissue was thawed 114 . the tissue was histologically tested 116 . the tissue was also tested to establish the density of primordial follicles 118 . on the basis of primordial follicle density and the number of pieces available , it is possible to estimate how many follicles remain , and how long they could potentially last . under local anesthesia , the ovarian cortical pieces were implanted 120 in a heterotopic location beneath the skin of the patient &# 39 ; s lower abdominal wall using a suture pull - through technique . first , a free end of a suture was threaded onto a half - circle cutting needle with a chord length ranging from 25 to 38 mm . next , the needle was inserted into a subcutaneous pocket . the needle was then passed through the skin , and the cortical piece was wedged into the subcutaneous pocket by pulling on the suture . additional details regarding the suture pull - through technique may be found in the technique of ovarian transplantation , described above . the ovarian cortical pieces may alternately be implanted in other heterotopic locations , such as subcutaneously in the forearm . although requiring ivf to fertilize any subsequently retrieved oocytes , a heterotopic site may be preferable to an orthotopic site because the technique does not require general anesthesia or abdominal surgery . further , the ovarian tissue may be closely monitored . if there is a cancer recurrence or cyst formation , for example , ovarian removal is easily accomplished from heterotopic locations . however , oocyte quality may be affected because of differences in temperature and blood flow to a heterotopic subcutaneous environment compared with an orthotopic pelvic location . before every cycle of ovarian stimulation 132 , ovarian suppression 130 was first performed by administering gonadotropin - releasing hormone antagonist or agonist ( gnrh ). ovarian stimulation 134 was then performed with a combination of follicle stimulating hormone ( fsh ) and human menopausal gonadotropins ( hmg ). oocyte maturity 136 was triggered by administering 250 μg of recombinant human chorionic gonadotropin ( hcg ), such as ovidrel ®, available from serono , located in norwell , mass ., usa . oocyte retrieval 138 was performed thirty - six to forty hours after triggering oocyte maturity 136 using a modified aspiration needle 10 , as seen in fig2 . as seen in fig3 and 4 , an ultrasonic probe 80 may be used to provide imaging . using this imaging technique , aspiration needle 10 may be used to pierce an individual follicle on an ovarian cortical piece and withdraw a single oocyte up through the aspiration needle 10 . the aspiration needle 10 , preferably with echogenic region 23 extending to the beveled tip 24 , was inserted into a patient 90 at a heterotopic location beneath the skin of the patient &# 39 ; s lower abdominal wall 92 . aspiration needle 10 was sonically imaged using an imaging device 82 with probe 80 to create image 84 . the aspiration needle 10 is made up of a needle cannula 20 , a handle 40 , an aspiration line 30 , a bung or stopper 50 , and a vacuum line 60 . the needle cannula 20 has an echogenic region 22 with a beveled tip 24 . echogenic region 22 incorporates components that are similar in design and / or function as described in u . s . pat . no . 5 , 081 , 997 , issued jan . 21 , 1992 and entitled echogenic devices , materials , and method . the contents of this patent are hereby incorporated by reference to avoid unnecessary duplication of the description of similar components . the aspiration needle 10 is modified from the echotip ® ovum aspiration needle , available from cook ®, cook urological inc ., spencer , ind ., usa . however , due to the heterotopic location of the implanted ovarian cortical pieces and the smaller volumes of the immature follicles , the needle cannula 20 and aspiration line 30 are reduced in size . the needle cannula 20 is preferably made from 19 gauge 304 stainless steel thinwall tubing , with an overall length of 4 cm , an outer diameter of 0 . 042 inches , and an inner diameter of 0 . 032 inches . however , needle lengths from 3 cm to 9 cm may also be used . in addition , other biocompatible materials , such as polycarbonates , may be used . moreover , other tubing gauges or thicknesses may also be used . preferably , the tubing is small enough to allow for accurate needle placement and oocyte retrieval , yet large enough to avoid needle flexibility . for example , tubing from 17 to 20 gauge may be used . the aspiration line 30 is preferably made from a 30 cm length of translucent 19 gauge tfe ( teflon ™) tubing , although other biocompatible materials and sizes may also be used . for example , aspiration line 30 may have a length from 15 to 60 cm , and a tubing gauge sized to match needle cannula 20 . handle 40 couples needle cannula 20 with one end of aspiration line 30 . handle 40 also provides a gripping surface to precisely manipulate aspiration needle 10 . the other end of aspiration line 30 passes through stopper 50 and terminates in a luer lock connection . stopper 50 is a formed as a silicone plug and is adapted to provide an air - tight seal for a collection tube 51 . vacuum line 60 also extends through stopper 50 . a vacuum source 52 may be attached to a luer lock connection on vacuum line 60 . vacuum source 52 provides a means for withdrawing an oocyte from a patient &# 39 ; s follicle and depositing the oocyte in the collection tube 51 attached to stopper 50 . due to the reduced size of aspiration needle 10 , a smaller vacuum pressure is needed than that used for standard needles designed for orthotopic aspiration . for example , a volume flow rate of 10 - 30 ml / minute with a vacuum pressure of 60 - 80 mm hg may be used for aspiration needle 10 . in contrast , for standard aspiration needles , volume flow rates of 30 - 40 ml / minute with vacuum pressures of 110 - 130 mm hg may be required . progesterone supplements were administered during every cycle , following oocyte retrieval . after oocytes were retrieved using aspiration needle 10 , they were evaluated to determine their maturity 140 . immature oocytes may be matured in vitro 142 . the oocyte was matured and fertilized in a complex , non - commercial sequential culture medium . one hypothesis predicts that aspiration of immature oocytes from smaller follicles followed by in vitro maturation may be the preferred approach to preserve the competence to undergo fertilization . further , oocyte maturity at heterotopic locations appears to be attained at 10 - 11 mm diameter , in contrast with a 16 - 17 mm diameter in orthotopic ovaries . after the oocyte is matured , an ivf technique 144 such as intracytoplasmic sperm injection technique ( icsi ) may be used to fertilize the oocyte , which will develop into an embryo . the oocyte and embryo was matured and cultured in this medium until about 48 hours after the injection . the embryo may then be evaluated to determine morphology , such as by a preimplantation genetic diagnosis by fluorescence and in - situ hybridization . normal embryos are then transferred to a patient &# 39 ; s uterus . fig5 illustrates the patient &# 39 ; s resumption of ovarian function afer ovarian transplantation . the peak oestradiol concentration accords with that of a typical cycle and is accompanied by a luteinising hormone ( lh ) surge , but no fsh increase is seen . the results of the patient &# 39 ; s percutaneous oocyte retrievals are shown in fig6 . fig6 is a graphical depiction of the stimulation type , peak oestradiol concentration , and follicle sizes on day of human chorionic gonadotropin administration and oocyte retrieval outcome from ovarian transplant . eight consecutive cycles of retrievals were conducted over eight months . of the approximately 20 oocytes obtained from the patient &# 39 ; s transplanted tissue , eight were suitable for ivf with her husband &# 39 ; s sperm . three of the eight oocytes were mature at retrieval , while the other five oocytes had to be matured in vitro . although the mature oocytes did not fertilize , two of the oocytes that were matured in vitro were fertilized via icsi . one embryo showed abnormal morphology and its growth was halted at the three - cell stage . the other embryo was transferred to the patient &# 39 ; s uterus . while the invention has been described with reference to details of the illustrated embodiment , these details are not intended to limit the scope of the invention as defined in the appended claims .
a method for aspirating an oocyte from a human female is provided . the method includes the step of providing at least one ovarian cortical piece , implanting the ovarian cortical piece in the human female at a heterotopic location , and triggering oocyte maturity in the at least one ovarian cortical piece . the method also includes the steps of providing an aspiration needle and retrieving at least one oocyte from the at least one ovarian cortical piece using the aspiration needle .