FSP or IUTPI?
Fallopian tube Sperm Perfusion  or Intrautero Tubo Peritoneal  Insemination: a prospective randomized study.

Leonidas Mamas M.D., Ph.D.
Neogenesis IVF Centre, 3 Kifisias Ave, 151 23 Marousi, Athens, Greece
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Published in Fertility and Sterility Journa, March 2006 (www.fertstert.org)

Ten (10) ml of inseminate used for Intrautero Tubo Peritoneal Insemination (IUTPI), an easy, non  invasive technique, give higher pregnancy rates than Fallopian tube Sperm Perfusion (FSP), with 4ml, in cases of unexplained infertility or male subfertility.

Objective: To evaluate the results of two different methods of insemination; Fallopian tube Sperm Perfusion (FSP) with 4 ml of inseminate and Intra Utero Tubo Peritoneal Insemination (IUTPI) with 10 ml of inseminate.
Design: Prospective randomised clinical study.
Setting: Private infertility centre.
Patients: Two hundred seventy six (276) couples, undergoing 403 cycles, with unexplained infertility, either mild, or moderate, male subfertility, mild or moderate endometriosis.
Interventions: Patients were assigned randomly to either FSP (Group A, n=138) treatment or IUTPI (Group B, n=138) treatment, with sealed envelopes. Both Groups followed the same mild ovarian stimulation protocol.  
Main outcome measures: Ninety-five overall pregnancies; thirty-five in Group A (FSP) and sixty in Group B (IUTPI).
Results: The pregnancy rate per cycle (presence of gestational sac with heart beats) was 17.6% in Group A (n=199) and 29.4% in Group B (n=204). These differences were statistically significant (P<.007). The odds ratio of getting pregnant, per patient, in Group B was 2.26%, 95% CI (1.36-3.77) compared to Group A.

Conclusions: The results of this study indicate that IUTPI may prove a useful technique in the treatment of unexplained infertility, mild or moderate male subfertility and mild or moderate endometriosis. (Three attempts of IUTPI may be beneficial before moving on to more invasive and expensive methods of assisted reproduction techniques).

Key words: Fallopian tube Sperm Perfusion, Intrautero Tubo Peritoneal Insemination, Intrauterine Insemination.

    The method of Fallopian tube Sperm Perfusion (FSP) was firstly described in 1992, by Kahn et al (1). This method involved the use of 4 ml. of inseminate, a considerably higher volume to the 0.5 ml previously used for  Intrauterine Insemination (IUI). Leakage of the inseminate was avoided by clamping the cervix and the pressure built in the uterine cavity lead to utero – tubal flushing. Consequently, the number of motile spermatozoa was increased in the female genital tract   overcoming, thus, low sperm count, suboptimal spermatozoa or total absence of spermatozoa at the site of fertilization. The results were encouraging and were confirmed in two more publications  (2, 3).

   It was observed that  the number  of  spermatozoa distributed within the Fallopian tubes around ovulation after IUI was low. Only a median number of 251 spermatozoa were recovered by flushing the tubes (4), and there was only a 49% chance of  peritoneal spermatozoa to be found, even when all semen characteristics were normal (5).
Ripps et al. (6) showed that, although the number of peritoneal spermatozoa recovered at laparoscopy after IUI was very low, their number increased after utero-tubal flushes. In addition, utero-tubal flushes required a certain pressure for the achievement of intrauterine perfusion  and spill  in normal tubes or in tubes with minimal adhesions  (7), because, in the mid follicular phase and before ovulation, the endometrial glandular lumen of the fallopian tubes were narrowed and contained quantities of granular and membranous material, as well as  increasing amounts of cytoplasmic debris, cellular fragments  (8) and tubal ostium membranes (9)
   In 1993, Li TC (10) described another  technique for FSP with good results, by using a Foley size 8 paediatric catheter with the balloon inflated in the uterine cavity to seal the uterocervical junction.Fanchin et al.(11) proposed the FAST System, for FSP with pregnancy results twice as high as standard IUI. The author of this article presented the use of the, specially designed for FSP, Double Nut Bivalve (DNB) Speculum  with modified tips which firmly clamp the cervix and the cervical canal, preventing any leakage of the inseminate leading to a higher fallopian sperm perfusion  with results twice as promising as standard IUI  (12).
   Observations in our clinical practice showed that, occasionally,  the 4 ml of inseminate used for FSP was not sufficient to overcome the tubal ostia, because the intrauterine pressure caused by this volume was not adequate.  Thus,  a larger volume of inseminate was gradually  used, ultimately reaching 10ml, while simultaneously checking the intrauterine pressure. There was an unexpected increase of pregnancy rates when a larger volume of inseminate was used.  Besides, 10 ml of contrast medium  is also used  in hysterosalpingography (HSG) (13). In addition, the possible therapeutic effect of HSG has been extensively discussed in various articles over the last decades, since the rate of live births increases after HSG, especially in patients with unexplained infertility, therefore, supporting  the hypothesis that  tubal “plugs” may be involved in proximal tubal blockage (14 , 15).      
   The volume of 10 ml of inseminate was sufficient enough to fill the uterine cavity, pass through the interstitial part of the tubes and the ampulla, finally reaching the peritoneal cavity and the Pouch of Douglas where it would be mixed with the peritoneal and follicular fluids. In this way Intra Utero Tubo Peritoneal Insemination (IUTPI) was achieved.
   This prospective randomized study was designed to compare the results of FSP and IUTPI.

MATERIALS  AND  METHODS
Patients  selection
   During the 30- month period from July 1, 2002, to Decemder 31, 2004, two hundred and seventy six (276) couples  undergoing  403 cycles, were treated  in our IVF Centre. Couples were  asked whether they would like to participate in this study. All couples suffered from unexplained infertility,  either mild or moderate,  male subfertility and mild or moderate endometriosis after treatment  (16). All women had a regular menstrual cycle of 25-33 days. Spontaneous ovulation was checked by vaginal ultrasound and normal serum progesterone concentrations by midluteal phase serum (P > 10 ng/ml). Serum levels of follicle stimulating hormone (FSH) <10 U/L, on day 3, indicated adequate ovarian reserve, whereas, luteinizing hormone (LH), prolactin, testosterone, sex hormone-binding globulin, thyroid hormone concentration had to be in the normal range. Chlamydia detection tests were also performed.  The body mass index had to be between 20 and 29 kg/m².  Men were asked to have a semen analysis and in cases of subfertility the test was repeated. Semen analysis was evaluated according to the World Health Organization criteria (17), the sperm morphology was assessed using strict criteria (18) and the Inseminate Motile sperm Count (IMC) recovered after gradients had to be more than 1´106.
   Two hundred thirty one (231) patients had primary infertility and 45 secondary. The mean duration of  infertility was 3.06 ± 1.22 years (1-6) for group A (FSP) and 3.15 ± 1.11 years (2-6) for group B (IUTPI). All patients had undergone  hysterosalpingography in the last 12 months and had had normal uterine cavity and bilateral tubal patency (19 , 20).  Thirty six of them had undergone laparoscopy because of a previous history suspicious of tubal disease. Patients had to be younger than 40 years old.

Controlled ovarian stimulation
   All patients underwent the same controlled ovarian stimulation protocol. The protocol consisted of clomiphene citrate followed by highly purified urinary FSH – hpuFSH -   (Altermon, IBSA Switzerland) or recFSH, (Puregon, Organon, The Netherlands or Gonal-f, Serono, Switzerland). 100 mg of  clomiphene citrate were administered daily from day 2 to 6 of the cycle, For patients with body mass index between 20 and 24kg/m2, clomiphene was followed by 75 IU of either hpuFSH, in 264 cycles, or 75 IU recFSH, in 60 cycles, from day 6 – 9. For patients with body mass index between 25 and 29 kg/m2, clomiphene was followed by 150 IU of either hpuFSH, in 64 cycles, or 75 IU recFSH, in 15 cycles, from day 6 – 9. A transvaginal ultrasonography was performed on day 2 of the cycle (check scan) which was repeated on the tenth day for the assessment of the number and diameter of the follicles, as well as  the thickness and the morphology of the endometrium. The serum oestradiol (E2) was also measured on that day. The administration of hpuFSH or recFSH was continued to the tenth day or, in some cases,  to the eleventh or twelveth day. 5000 IU of  Human chorionic gonadotropin (HCG, Pregnyl, Organon) were administered  when the leading follicle was 18-19 mm in diameter and 2-3 follicles of 16-17 mm were also present. In addition, the transvaginal power Doppler utrasonography should show high-grade perifollicular vascularity (21). The treatment cycles were cancelled  when serum oestradiol concentration exceeded 1000pg/ml or more and  four follicles or more had matured, for the risk of multiple gestation (22, 23, 24). Insemination was performed 36-40 h after HCG administration.
Randomization
   Women were allocated randomly in Groups A (FSP), or B (IUTPI) on the day of HCG administration with sealed envelopes  and remained in the same group throughout  all attempts.
     

Sperm preparation  
   Semen was collected by masturbation into a sterile jar following 3 – 4 days of sexual abstinence. Semen analysis was evaluated after liquefaction according to the World Health Organization criteria (17), whereas, sperm morphology was assessed using strict criteria  (18).
A  two - layer gradient technique was used for sperm preparation. 1ml of 45% SpermGrad (Vitrolife, Sweden) + Ham’s F-10 (Gibgo BRL, Life Technologies, Scotland) was layered over 1ml 90% SpermGrad, in a 15 ml conical centrifuge tube. The undiluted semen was layered over this gradient and the tube was centrifuged for 20 minutes at 250g. The supernatant was discarded and the pellet diluted in 7 ml of Universal IVF medium (MediCult , Copenhagen, Denmark) and re-centrifuged for 8 min at 500g.
     The supernatant was discarded after re-centrifugation and the remaining final pellet was diluted in 4ml or 10 ml of  Universal IVF medium for  FSP and IUTPI respectively. All solutions were also  analysed to evaluate the  Inseminate Motile sperm Count (IMC).

Insemination
   Insemination was performed 36 - 40 hours after HCG administration in both groups.
FSP with 4 ml of inseminate was carried out using the cervical clamp Double Nut Bivalve Speculum (DNB Speculum® , Parthenon Medical Ltd, Athens, Greece), which was specially designed for clamping and sealing the cervix. The cervix and vaginal fornices were cleaned with a cotton swab and the external cervical os with antiseptic. The use of DNB Speculum®   has been described elsewhere (12).

   FSP was performed  slowly,  4ml/min, using a 18 mm insemination catheter (Wallace, UK) which  was atraumatically  inserted through the cervical canal into the upper part of the uterine cavity.  No leakage was usually observed. The speculum was kept locked, clamping the cervix for, at least, one or two minutes. Before removing the DNB Speculum®, the DNB test was performed by pulling  the piston of the syringe in order to check  the amount of the inseminate remaining in the uterine cavity.  If it was more than 1 ml, insemination was repeated.  The DNB Speculum® was removed only when the inseminate retrieved  was less than 1 ml.
   IUTPI with 10 ml of inseminate was also carried out using the cervical clamp DNB Speculum®. Insemination was performed slowly in 1 – 2min. while, simultaneously checking  the intrauterine pressure with a Schulze manometer (Germany) used in hysterosalpingography. The lowest pressure checked was 80 mm/Hg, the highest 240 mm/Hg and the mean pressure 140 mm/Hg. The pressure gradually builting up in the uterine cavity,  allowed the inseminate  to overcome the tubal  ostia and by flushing the Fallopian tubes reach the pouch of Douglas. Intraperitoneal insemination was thus achieved and the number of spermatozoa was increased throughout all the genital  tract and the Pouch of Douglas. The speculum was kept locked, clamping the cervix for one more minute at least. The DNB test was performed as described above. One insemination was performed per cycle and no infection was observed.    
Luteal  support
   The luteal phase was supported  by oral supplementation of 300 mg micronized progesterone (Utrogestan  Laboratories Besins Incovesco, Paris, France).    

Statistical Analysis
   The primary aim of this study was to compare the overall pregnancy rate per Group. Based on interim results and existing data, we assumed that FSP treatment would yield a pregnancy rate of 20%. Sample size calculation showed that a 15% increase of the overall pregnancy rate could be detected between the two groups with a power of 80% using a χ2 test. Pregnancy rates per cycle were also calculated and compared by a χ2 test at a=0.05.
   Baseline characteristics of women in the two groups were presented and their mean values were compared by independent samples t-test at a= 0.05. Clinical pregnancy was defined by the presence of foetal heart beat detected by ultrasound examination.
Results
   Of the 276 couples enrolled in this study, 138 were allocated in  Group A (FSP), and 138 in Group B (IUTPI). All patients  underwent a total of 403 treatment cycles and were randomized  in two treatment groups:  Group A (n=199): FSP with 4 ml of sperm inseminated and Group B (n=204): IUTPI with 10 ml of sperm inseminated.
   As far as the baseline characteristics of the patients of both groups are concerned, there was no statistical difference in the age, the number of mature follicles (> 17 mm of diameter ),  serum E2 levels,  the thickness of endometrium as measured by vaginal ultrasound on the day of randomization (day of HCG administration) and the Inseminate Motile sperm Count (IMC), obtained after preparation (Table 1).

Table 1.
Baseline characteristics of the study groups.

   In both treatment groups, 95 women got pregnant, yielding an overall pregnancy rate of 34.4%. Considering the total number of achieved pregnancies, we observed that 35/138 (25.4%) pregnancies had been achieved in group A,  and  60/138 (43.5%) pregnancies in group B (p-value= 0.002). The odds ratio of getting pregnant in group B compared to group A is 2.26, 95% CI (1.36-3.77). The total rate of pregnancies per cycle in group A was 17.6% and in group B was 29.4%  (p-value=0.007) (Table 2).

Table 2
Number of pregnancies and corresponding rate per cycle in the two groups.
 

   Detailed information about the pregnancy rates in each cycle for the two groups is presented in table 2. Difference on rates of pregnancy in the first cycle is statistically significant (p= 0.012), while in the second and third cycle differences are not statistically significant, although IUTPI method keeps yielding higher pregnancy rates (p= 0.236, p= 0.693). Figure 1 illustrates the above mentioned results.
Four missed abortion occurred among the patients in Group A (FSP) and five in Group B (IUTPI).  Three twin pregnancies in Group A (FSP) and five in Group B (IUTPI) as well as a set of quadruplets, which was reduced to twin pregnancy. Three cases of mild ovarian hyperstimulation syndrome (OHSS) occurred in all groups (25). No case of moderate or severe OHSS was observed.

Discussion

   Interest in Intrauterine Insemination (IUI) as well as Fallopian tube Sperm Perfusion (FSP) and other similar techniques,  was renewed by specialists, when new sperm preparation methods were developed  for In vitro Fertilization (IVF). These procedures improve the fertilizing ability of the sperm in vivo and in vitro by removing most adverse agents such as seminal plasma, prostaglandins,  bacteria, white blood cells,  cellular debris, immature germ cells, dead, damage and abnormal spermatozoa which produce free oxygen radicals (26, 27, 28)    
   This prospective, randomized study presents the encouraging results of IUTPI (inseminate volume 10 ml) compared with FSP (4 ml inseminate) in cases with unexplained infertility, mild and moderate male factor infertility and mild or moderate endometriosis. The pregnancy rates per cycle were 17.6% for the FSP Group (Group Α) and 29.4% for the IUTPI Group (Group Β). Βoth Groups followed the same mild ovarian stimulation protocol which was cost-effective, well tolerated, and required less injections per cycle (29). There was no statistically significant difference regarding the age of the patients treated, the mean number of follicles, serum oestradiol, endometrial thickness on the day of HCG administration and the  Inseminate Motile sperm Count (IMC). All cases had one insemination per cycle (30, 31).
   Fallopian tube sperm perfusion (FSP) with  controlled ovarian stimulation  was first described by Khan et al in 1992  showing encouraging results for cases  of unexplained infertility, with pregnancy rates ranging from 24.1% - 40.0 %  (1, 2, 3, 10, 11, 12). Although there are a few articles which report that FSP produces no statistically significant results compared to standard IUI in cases of unexplained infertility  (32, 33, 34, 35, 36), two meta-analyses done in 1999 (37), and 2004  (38) showed that FSP is beneficial in cases of unexplained infertility after ovarian stimulation. Ricci et al., 2001(39) confirms the good results of FSP for unexplained infertility, whereas Strandell et al., 2003 (40) showed that pregnancy results after FSP were not impaired by lower  sperm count and morphology. The wide range of the results given by FSP enthusiasts may be due to the different instruments used in order to facilitate the method (Alis clamp, Foley catheter, FAST System, DNB Speculum, ZUI catheter, Hysterosalpingography device). The DNB Speculum was used in this study.
   With FSP the inseminate perfuses the fallopian tubes and even reaches the pouch of Douglas. The higher pregnancy  rates of FSP  may be due to the higher concentration of spermatozoa  at the sites of fertilization.
   IUTPI resulted from observations made while FSP was under way which showed that the amount of 4ml of inseminate was not always adequate to increase the intrauterine pressure enough  to help the inseminate be pushed through the tubal ostia. So the volume of inseminate was gradually  increased, while simultaneously checking the intrauterine pressure. When the 10ml were finally reached the pregnancy rates showed an unexpected improvement. It should be noted here that 10 ml of contrast medium  is also used  in hysterosalpingography (13) and the possible therapeutic effect of YSG has been extensively discussed since the rate of live births increases after YSG, especially in patients with unexplained infertility, supporting,  therefore, the hypothesis that  tubal “plugs” may be involved in proximal tubal blockage, regardless of the contrast material, oil or water, used (14,15).   
   In this study, the intrauterine pressure was checked with a manometer and ranged from 80 mm/Hg to  240 mm/Hg with mean reading 140 mm/Hg. Baker and Adamson (7) gave lower readings probably because they measured the intrauterine pressure in the mid follicular phase, whereas, in this study, intrauterine pressure was checked around ovulation when the endometrial glandural lumen of the interstitial part of the Fallopian tube is narrowed and contains quantities of granular and membranous material, as well as  amounts of cytoplasmic debris and cellular fragments (8). The highest pressure was observed when 3 – 4 ml of sperm was inseminated which caused the tubal ostia to open. As soon as the inseminate passed through the tubal ostia and into the Fallopian tubes the pressure, according to the manometer, dropped to 10-30 mm/Hg. In addition, the opening of the tubal ostia could also be detected by the decrease of the syringe piston’s resistance.
   The volume of 10 ml of inseminate used in IUTPI was sufficient enough to fill the uterine cavity and, through the Fallopian tubes, reach  the peritoneal cavity and the Pouch of Douglas where it would be mixed with the peritoneal and follicular fluids, thus, accomplishing a non invasive intraperitoneal insemination, by flushing the tubes.
   Guidi et al(41) showed that  peritoneal fluids from hormonally-stimulated cycles, sustained sperm motility better than spontaneous cycles, while Revelli et al (42) showed that Follicular fluid  in hormonally-stimulated cycles always positively influence progressive motility of the sperm while sustaining  the number of motile spermatozoa.  This fact explains the good results of intraperitoneal insemination (IPI) in humans with very low sperm count  (42, 44, 45, 46)  as well as in mammals (46) which become even better when IPI is combined with IUI (47).
   However, because IPI is a more invasive technique, it should be used when other methods are difficult to perform, as in the presence of a tight cervical canal (48, 49).
    The excellent results of IUTPI (29,4%) may be due to the better flushing of the Fallopian tubes obtained by the 10ml of inseminate performing thus a non invasive IPI, to the increase of the number of spermatozoa in the peritoneal and follicular fluids and, finally,  to the increase of the number of spermatozoa at the sites of fertilization.     
   In conclusion, IUTPI may prove a useful technique in the treatment of unexplained infertility, mild or moderate male subfertilty and mild or moderate endometriosis. Three attempts of IUTPI may be beneficial before moving on to more invasive and expensive methods of assisted reproduction techniques.  

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